CN101247825B - Dr5 antibodies and uses thereof - Google Patents

Abstract

The invention concerns anti-DR5 antibodies with improved properties, compositions comprising such antibodies, methods and means for making such antibodies, and their therapeutic use, in particular in the treatment of cancer.

Description

DR5 antibody and uses thereof

Invention field

The present invention relates generally to DR5 antibody, comprises agonistic antibody, and uses the method for this type of DR5 antibody.

Background of invention

Multiple ligands and the receptor that belongs to tumor necrosis factor (TNF) superfamily identified in this area.Tumor necrosis factor-alpha (" TNF-α ") is arranged in these parts, tumor necrosis factor-β (" TNF-β " or " Lymphotoxin-α "), lymphotoxin-β (" LT-β "), the CD30 part, the CD27 part, CD40L, the OX-40 part, the 4-1BB part, LIGHT, Apo-1 part (also referred to as FasL or CD95 part), Apo2L/TRAIL (also referred to as Apo2L or TRAIL), Apo-3 part (also referred to as TWEAK), APRIL, the OPG part is (also referred to as the RANK part, ODF or TRANCE) and TALL-1 (also referred to as BlyS, BAFF or THANK) (referring to for example Ashkenazi, Nature Review, 2:420-430 (2002), Ashkenazi and Dixit, Science, 281:1305-1308 (1998), Ashkenazi andDixit, Curr.Opin.Cell Biol., 11:255-260 (2000), Golstein, Curr.Biol., 7:750-753 (1997), Wallach, Cytokine Reference, Academic Press, 2000, pages377-411, Locksley et al., Cell, 104:487-501 (2001), Gruss and Dower, Blood, 85:3378-3404 (1995), Schmid et al., Proc.Natl.Acad.Sci., 83:1881 (1986), Dealtryet al., Eur.J.Immunol., 17:689 (1987), Pitti et al., J.Biol.Chem., 271:12687-12690 (1996), Wiley et al., Immunity, 3:673-682 (1995), Browning et al., Cell, 72:847-856 (1993), Armitage et al., Nature, 357:80-82 (1992), WO97/01633, be disclosed on January 16th, 1997, WO97/25428, be disclosed on July 17th, 1997, Marsters et al., Curr.Biol., 8:525-528 (1998), Chicheportiche et al., Biol.Chem., 272:32401-32410 (1997), Hahne et al., J.Exp.Med., 188:1185-1190 (1998), WO98/28426, be disclosed on July 2nd, 1998, WO98/46751, be disclosed on October 22nd, 1998, WO98/18921, be disclosed on May 7th, 1998, Moore et al., Science, 285:260-263 (1999), Shu et al., J.Leukocyte Biol., 65:680 (1999), Schneider etal., J.Exp.Med., 189:1747-1756 (1999), Mukhopadhyay et al., J.Biol.Chem., 274:15978-15981 (1999)).

By these TNF families, ligand-mediated various kinds of cell is replied brings out normally and to start by them and specific cells receptors bind.Some but non-all TNF family's ligand binding cell surfaces " death receptor ", and bring out thus the various biological activity to activate enzyme (the Salvesen et al. of Caspase or execution cell death or apoptosis pathway, Cell, 91:443-446 (1997)).Comprise that the TNF receptor superfamily member who has identified up to now TNFR1, TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also referred to as Apo-1 or CD95), DR4 (also referred to as TRAIL-R1), DR5 (also referred to as Apo-2 or TRAIL-R2), DcR1, DcR2, OPG (OPG), RANK and Apo-3 (also referred to as DR3 or TRAMP) are (referring to for example Ashkenazi, Nature Reviews, 2:420-430 (2002); Ashkenazi and Dixit, Science, 281:1305-1308 (1998); Ashkenazi and Dixit, Ctrr.Opin.Cell Biol., 11:255-260 (2000); Golstein, Curr.Biol., 7:750-753 (1997); Wallach, Cytokine Reference, Academic Press, 2000, pages377-411; Locksley et al., Cell, 104:487-501 (2001); Gruss and Dower, Blood, 85:3378-3404 (1995); Hohman et al., J.Biol.Chem., 264:14927-14934 (1989); Brockhaus et al., Proc.Natl.Acad.Sci., 87:3127-3131 (1990); EP417,563, be disclosed on March 20th, 1991; Loetscher et al., Cell, 61:351 (1990); Schall et al., Cell, 61:361 (1990); Smith et al., Science, 248; 1019-1023 (1990); Lewis et al., Proc.Natl.Acad.Sci., 88:2830-2834 (1991); Goodwin et al., Mol.Cell.Biol., 11:3020-3026 (1991); Stamenkovic et al., EMBO J., 8:1403-1410 (1989); Mallett et al., EMBO J., 9:1063-1068 (1990); Anderson et al., Nature, 390:175-179 (1997); Chicheportiche et al., J.Biol.Chem., 272:32401-32410 (1997); Pan et al., Science, 276:111-113 (1997); Pan et al., Science, 277:815-818 (1997); Sheridan et al., Science, 277:818-821 (1997); Degli-Esposti et al., J.Exp.Med., 186:1165-1170 (1997); Marsters et al., Curr.Biol., 7:1003-1006 (1997); Tsuda et al., BBRC, 234:137-142 (1997); Nocentiniet al., Proc.Natl.Acad.Sci., 94:6216-6221 (1997); VonBulow et al., Science, 278:138-141 (1997)).

These TNF receptor family members of great majority share the typical structure of cell surface receptor, comprise extracellular region, cross-film district and intracellular region, and other member natural discovery is the soluble protein that lacks cross-film and born of the same parents' intracellular domain.Born of the same parents' outer part of typical case TNFR contains from NH ₂a plurality of repetition aminoacid sequences that are rich in cysteine structure territory (CRD) that-end is initial.

Before several years, the part that will be called Apo2L or TRAIL be accredited as the member of TNF cytokine family (referring to for example Wiley et al., Immunity, 3:673-682 (1995); Pitti et al., J.Biol.Chem., 271:12697-12690 (1996); WO97/01633; WO97/25428; United States Patent (USP) 5,763,223, be issued on June 9th, 1998; United States Patent (USP) 6,284,236, be issued to calendar year 2001 JIUYUE 4 days).Total length native sequences people Apo2L/TRAIL polypeptide is 281 amino acid whose II type transmembrane proteins.The extracellular region that some cell can cut polypeptide by enzymatic becomes natural soluble form polypeptide (Mariani et al., J.Cell.Biol., 137:221-229 (1997)) next life.The Crystallographic Study of soluble form Apo2L/TRAIL has been disclosed to homotrimer structure (Hymowitz et al., Molec.Cell, the 4:563-571 (1999) with TNF and other related protein similar; Cha et al., Immunity, 11:253-261 (1999); Mongkolsapaya et al., Nature Structural Biology, 6:1048 (1999); Hymowitz et al., Biochemistry, 39:633-644 (2000)).But, different from other TNF family member, find that Apo2L/TRAIL has unique architectural feature, three cysteine residues (in homotrimer the 230th of each subunit the) together with the zinc atom coordination, and zinc is in conjunction with being that important (Hymowitz et al., see above for trimer stability and biologic activity; Bodmer etal., J.Biol.Chem., 275:20632-20637 (2000)).

In document, have been reported, Apo2L/TRAIL may work in immune adjusting, comprises autoimmune disease, such as rheumatoid arthritis (referring to for example Thomas et al., J.Immunol., 161:2195-2200 (1998); Johnsen et al., Cytokine, 11:664-672 (1999); Griffith etal., J.Exp.Med., 189:1343-1353 (1999); Song et al., J.Exp.Med., 191:1095-1103 (2000)).

Also has report, the Apo2L/TRAIL of soluble form is apoptosis-induced in multiple cancerous cell, comprise colon, lung, breast, prostate, bladder, kidney, ovary and the cerebral tumor, and melanoma, leukemia and multiple myeloma (referring to for example Wiley et al., see above; Pitti et al., see above; United States Patent (USP) 6,030,945, be issued on February 29th, 2000; United States Patent (USP) 6,746,668, be issued on June 8th, 2004; Rieger et al., FEBS Letters, 427:124-128 (1998); Ashkenazi et al., J.Clin.Invest., 104:155-162 (1999); Walczak et al., Nature Med., 5:157-163 (1999); Keane et al., Cancer Research, 59:734-741 (1999); Mizutani et al., Clin.Cancer Res., 5:2605-2612 (1999); Gazitt, Leukemia, 13:1817-1824 (1999); Yuet al., Cancer Res., 60:2384-2389 (2000); Chinnaiyan et al., Proc.Natl.Acad.Sci., 97:1754-1759 (2000)).In the body of Mus tumor model, research also shows, uses separately Apo2L/TRAIL or can bring into play substantial antitumor action with chemotherapy or chemotherapy combined radiotherapy (referring to for example Ashkenazi et al., to see above; Walzcak et al., see above; Gliniak et al., Cancer Res., 59:6153-6158 (1999); Chinnaiyan et al., see above; Roth et al., Biochem.Biophys.Res.Comm., 265:1999 (1999); PCT applies for US/00/15512; PCT applies for US/01/23691).With being permitted, eurypalynous cancerous cell is contrary, and most of normal cell type lists reveal the apoptosis that some recombinant forms Apo2L/TRAIL is induced to be had resistance (Ashkenazi et al., see above; Walzcak et al., see above).The people such as Jo report, the soluble form Apo2L/TRAIL of polyhistidine labelling is apoptosis-induced in the normal liver cell who separates in vitro, quite different (Jo et al., Nature Med., the 6:564-567 (2000) in inhuman source; Nagata, Nature Med., 6:502-503 (2000)).Think, some restructuring Apo2L/TRAIL prepared product can be different to diseased cells and normal cell aspect biochemical characteristic and biologic activity, this existence of depending on tag molecule for example whether, zinc content and trimer percentage composition be (referring to Lawrence et al., Nature Med., Letter tothe Editor, 7:383-385 (2001); Qin et al., Nature Med., Letter to the Editor, 7:385-386 (2001)).

Have been found that Apo2L/TRAIL is in conjunction with at least five kinds of isoacceptors not.At least two kinds of receptors in conjunction with Apo2L/TRAIL comprise functional Matrix cell death domain.A kind of such receptor is called " DR4 " (or being called TR4 or TRAIL-R1) (Pan et al., Science, 276:111-113 (1997); WO98/32856, be disclosed on July 30th, 1998; WO99/37684, be disclosed on July 29th, 1999; WO00/73349, be disclosed in December in 2000 7; US6,433,147, be issued on August 13rd, 2002; US6,461,823, be issued on October 8th, 2002; US6,342,383, be issued on January 29th, 2002).

Another kind of such Apo2L/TRAIL receptor is called DR5 (or being called Apo-2, TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER) (referring to for example Sheridan et al., Science, 277:818-821 (1997); Pan et al., Science, 277:815-818 (1997); WO98/51793, be disclosed on November 19th, 1998; WO98/41629, be disclosed in JIUYUE in 1998 24; Screaton et al., Curr.Biol., 7:693-696 (1997); Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO98/35986, be disclosed on August 20th, 1998; EP870,827, be disclosed on October 14th, 1998; WO98/46643, be disclosed on October 22nd, 1998; WO99/02653, be disclosed on January 21st, 1999; WO99/09165, be disclosed on February 25th, 1999; WO99/11791, be disclosed on March 11st, 1999; US2002/0072091, be disclosed on August 13rd, 2002; US2002/0098550, be disclosed in calendar year 2001 December 7 days; US6,313,269, be issued to calendar year 2001 December 6 days; US2001/0010924, be disclosed in August 2 calendar year 2001; US2003/01255540, be disclosed on July 3rd, 2003; US2002/0160446, be disclosed on October 31st, 2002; US2002/0048785, be disclosed on April 25th, 2002; US6,342,369, be issued in February, 2002; US6,569,642, be issued on May 27th, 2003; US6,072,047, be issued on June 6th, 2000; US6,642,358, be issued on November 4th, 2003; US6,743,625, be issued on June 1st, 2004).As DR4, DR5 it is reported that comprising three in part outside its born of the same parents is rich in cysteine structure territory and single Matrix cell death domain and can sends apoptotic signal by ligand binding (or by the agonistic antibody equimolecular in conjunction with such as the simulation ligand activity).The crystal structure of the complex formed between Apo-2L/TRAIL and DR5 is described in Hymowitz et al., Molecular Cell, 4:563-571 (1999).

After ligand binding, DR4 and DR5 can independently cause apoptosis (Kischkel et al. via the adapter molecule of the death domain-containing that is called FADD/Mort1 by raising and activate apoptosis starting material Caspase-8, Immunity, 12:611-620 (2000); Sprick et al., Immunity, 12:599-609 (2000); Bodmer et al., Nature Cell Biol., 2:241-243 (2000)).Particularly, DR5 sends apoptotic signal by " extracellular is at (cell extrinsic) " approach, and it does not rely on p53 tumor suppressor gene (Ashkenazi and Dixit, Science281:1305-8 (1998); Ashkenazi, NatRev Cancer2:420-30 (2002)).The Matrix cell death domain place that the activation of this approach is involved in the receptor of activation forms and induces dead Signaling complex (DISC) rapidly.At first, adapter molecule FADD interacts in conjunction with DR5 (Kischkel et al., supra by having a liking for same sex death domain; Sprick et al., supra; Bodmer et al, supra).Then, FADD raises the protease that starts apoptosis, and Caspase-8 and Caspase-10 approach (proximity) and mediate their activation by inducing.Oneself's assembling is carried out in Caspase-9 and Caspase-10, and the active Caspase subunit of solubility is discharged in kytoplasm, and at this, they assemble and cut the effector Caspase, such as Caspase-3 and Caspase-7.Cutting causes the activation of effector Caspase, and they carry out apoptosis (apoprotix) programmed cell (Thomberry and Lazebnik, Science281:1312-6 (1998)).

Apo2L/TRAIL it is reported the receptor that also in conjunction with those, is called DcR1, DcR2 and OPG, think that the function of mortifier that these receptors performance signals transmit but not transducer is (referring to for example DCR1 (also referred to as TRID, LIT or TRAIL-R3) (Pan et al., Science, 276:111-113 (1997); Sheridan et al., Science, 277:818-821 (1997); McFarlane et al., J.Biol.Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters, 416:329-334 (1997); Degli-Esposti et al., J.Exp.Med., 186:1165-1170 (1997); Mongkolsapaya et al., J.Immunol., 160:3-6 (1998)); DCR2 (also referred to as TRUNDD or TRAIL-R4) (Marsters et al., Curr.Biol., 7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998); Degli-Esposti et al., Immunity, 7:813-820 (1997)); And OPG (Simonet et al., see above)).Contrary with DR4 and DR5, DcR1 and DcR2 receptor do not send apoptotic signal.

Reported the antibody of some and DR4 and/or DR5 receptors bind in document.For example, the anti-DR4 antibody that has excitement or apoptosis activity for the DR4 receptor and in some mammalian cell is described in for example WO99/37684, is disclosed on July 29th, 1999; WO00/73349, be disclosed on July 12nd, 2000; WO03/066661, be disclosed on August 14th, 2003.Also can be referring to for example Griffithet al., J.Immunol., 162:2597-2605 (1999); Chuntharapai et al., J.Immunol., 166:4891-4898 (2001); WO02/097033, be disclosed in December in 2002 2; WO03/042367, be disclosed on May 22nd, 2003; WO03/038043, be disclosed on May 8th, 2003; WO03/037913, be disclosed on May 8th, 2003.Equally describe some Anti-DR5 antibody, referring to for example WO98/51793, be disclosed on November 8th, 1998; Griffith et al., J.Immunol., 162:2597-2605 (1999); Ichikawa et al., Nature Med., 7:954-960 (2001); Hylander et al., " An Antibody to DR5 (TRAIL-Receptor2) Suppresses theGrowth of Patient Derived Gastrointestinal Tumors Grown in SCID mice ", Abstract, 2d International Congress on Monoclonal Antibodies in Cancers, Aug.29-Sept.1,2002, Banff, Alberta, Canada; WO03/038043, be disclosed on May 8th, 2003; WO03/037913, be disclosed on May 8th, 2003.In addition, described some and DR4 and DR5 receptor have all been there is to the antibody (referring to for example United States Patent (USP) 6,252,050, being issued to June 26 calendar year 2001) of cross reactivity.

Summary of the invention

The invention provides DR5 antibody, it can specific binding people DR5 and/or can the regulation and control biologic activity, particularly apoptosis relevant with DR5 and/or its part, thereby can be used for treating various diseases and pathology illness, comprises cancer or immune correlated disease.

On the one hand, the present invention pays close attention to Anti-DR5 antibody or its fragment, at least one place sudden change in the heavy chain that it comprises full length antibody 16E2 and/or light chain (being respectively SEQ ID NO:11 and 13), wherein said antibody or antibody fragment demonstrate at least identical with the antibody 16E2 affinity to DR5, and/or show at least identical with antibody 16E2 biologic activity and/or potential.In a specific embodiment, described antibody or antibody fragment will be with full length antibody 16E2 in conjunction with substantially the same epi-positions.In another embodiment, described Anti-DR5 antibody will show the affinity to DR5 higher than full length antibody 16E2, and/or demonstrate with respect to the biologic activity of full length antibody 16E2 rising and/or the potential of rising.In also having an embodiment, Anti-DR5 antibody of the present invention demonstrate at least identical with the strand Fc Anti-DR5 antibody 16E2 put down in writing in the WO98/51793 affinity to DR5 with antibody fragment and/or show at least with WO98/51793 in identical biologic activity and/or the potential of the strand Fc Anti-DR5 antibody 16E2 that puts down in writing.

In one embodiment, described Anti-DR5 antibody comprises and has alternative heavy chain and/or the light chain in arbitrary listed at least one place in table 1-7 and 9-12.

In another embodiment, described Anti-DR5 antibody comprises a place or the many places sudden change in 16E2 antibody heavy chain variable region framework.

In also having an embodiment, described DR5 antibody comprises the framework sudden change that is selected from lower group: Q6E, V11L, E12V, R13Q and K105Q.

In another embodiment, described Anti-DR5 antibody comprises following all framework sudden changes: Q6E, V11L, E12V, R13Q and K105Q.

In also having an embodiment, described Anti-DR5 antibody or its fragment comprise at least one place sudden change in full length antibody 16E2 heavy chain (SEQ ID NO:11).

In a different embodiment, described Anti-DR5 antibody or its fragment comprise at least one place sudden change that is selected from lower group: T28A, G33A, M34L, M34A, M34I, M34S, N53Q, N53Y and L102Y in aminoacid sequence SEQ ID NO:11.

In another embodiment, described Anti-DR5 antibody or its fragment comprise at least one place sudden change in G99A in aminoacid sequence SEQID NO:11 and R100A.

In also having an embodiment, described Anti-DR5 antibody or its fragment comprise the one group of sudden change that is selected from lower group: (i) N53Q in aminoacid sequence SEQ ID NO:11, L102Y; (ii) M34L, N53Q, L102Y; (iii) N53Y, L102Y; (iv) M34L, N53Y, L102Y; (v) G33A, N53Q, L102Y; (vi) M34L, N53Y, L102Y; (vii) G33A, N53Q, L102Y; (viii) G33A, N53Y, L102Y; (ix) T28A, N53Q, L102Y; Reach (x) T28A, N53Y, L102Y.

In another embodiment, described Anti-DR5 antibody or its fragment comprise at least one place sudden change in total length 16E2 light chain of antibody (SEQ ID NO:13).

In a specific embodiment, described light chain is the λ chain.

In another specific embodiment, described light chain sudden change is in CDR L1.

In another embodiment, described light chain sudden change is selected from lower group: Q24A, Q24S, G25A, D26E, S27A, L28A, R29A, S30A, Y31A, Y31K, Y32H, A33G, S34A and S34Y in aminoacid sequence SEQ ID NO:13.

In also having an embodiment, described light chain sudden change is selected from lower group: (i) Q24S in aminoacid sequence SEQ IDNO:13, D26E, Y31K, S34Y; Reach (ii) D26E, Y31K.

In a different embodiment, described light chain sudden change is in CDR L2.

Thereby for example, described sudden change can be selected from lower group: G50A, G50K, G50S, K51D, N52A, N52S, N52L, N52Q, N53A, N53E, N53Q, N53S, P55A and S56A in aminoacid sequence SEQ ID NO:13.

In another embodiment, described antibody can comprise the one group of sudden change that is selected from lower group: (i) G50K in aminoacid sequence SEQ ID NO:13, K52S, N53E; (ii) G50S, K51D, N52S, N53E; (iii) N52S, N53E; Reach (iv) N52Q, N53S.

In another embodiment, described light chain sudden change is in CDR L3.

In also having an embodiment, described antibody comprises at least one place sudden change that is selected from lower group: N89A, N89L, N89Q, R91A, S93A, N95aA, N95aT, N95aQ, H95bA, N95bY, V96A, V97A in aminoacid sequence SEQ ID NO:13.

Perhaps, described Anti-DR5 antibody can comprise the one group of sudden change that is selected from lower group: (i) N89L in aminoacid sequence SEQ IDNO:13, R91A, N95aT, H95bY; Reach (ii) N95aT, H95bY.

In another embodiment, described Anti-DR5 antibody comprises the one group of light chain sudden change that is selected from lower group: (i) Q24S in aminoacid sequence SEQ ID NO:13, G50K, K51D, H95bY; (ii) Q24S, K51A, D92S, S93Y; Reach (iii) Q24S, K51A, R91A, but also can comprise the one group of heavy chain sudden change that is selected from lower group: (i) M34L in aminoacid sequence SEQ ID NO:11, N53Q, L102Y; (ii) M34L, N53Y, L102Y; (iii) G33A, N53Q, L102Y; (iv) G33A, N53Y, L102Y; (v) M34L, N53Q, L102Y; (vi) M34L, N53Y, L103Y; (vii) G33A, N53Q, L102Y; (viii) G33A, N53Y, L102Y; Reach (ix) T28A, N53Q, L102Y, and the listed framing sudden change of option list 5.

In a specific embodiment, described Anti-DR5 antibody comprises following sudden change: Q24S, K51A, R91A in the G33A in sequence SEQ IDNO:11, N53Q, L102Y and sequence SEQ ID NO:13, but also can comprise the sudden change of at least one place framework, it can be at least one in the residue 6,11,12,13 and 105 in SEQ ID NO:11 for example.

In a specific embodiment, described Anti-DR5 antibody is selected from lower group: Apomab 1.1,2.1,3.1,4.1,5.1,6.1,7.1,8.1,9.1,1.2,2.2,3.2,4.2,5.2,6.2,7.2,8.2,9.2,1.3,2.3,3.3,4.3,5.3,6.3,7.3,8.3 and 9.3.

In a specific embodiment, described Anti-DR5 antibody is selected from lower group: Apomab 5.2,5.3,6.2,6.3,7.2,7.3,8.3 and 25.3.

In also having a specific embodiment, described Anti-DR5 antibody is Apomab 7.3 or Apomab 8.3, and especially Apomab 7.3.

In also having an embodiment, described Anti-DR5 antibody is antibody fragment, and it can be selected from lower group: Fab, Fab ', F (ab ') 2 and Fv fragment, double antibody, single-chain antibody molecule and the multi-specificity antibody formed by antibody fragment.

In other embodiments, described antibody can be single-chain antibody.

Described Anti-DR5 antibody can for example have active anticancer, such as for example they can have the ability that activates or stimulate apoptosis in cancerous cell.Described cancer comprises for example cancer, lymphoma, blastoma, sarcoma and leukemia.

The more specifically example of cancer comprises squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer (NSCLC), non-Hodgkin's (non-Hodgkin ' s) lymphoma, blastoma, human primary gastrointestinal cancers, renal carcinoma (renalcancer), ovarian cancer, liver cancer (liver cancer), gastric cancer, bladder cancer, hepatoma (hepatoma), breast carcinoma, colon cancer, colorectal carcinoma, cancer of pancreas, carcinoma of endometrium, salivary-gland carcinoma, renal carcinoma (kidneycancer), carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, hepatocarcinoma (hepatic carcinoma), and head and neck cancer.

Concrete cancer group comprises: pulmonary carcinoma, for example nonsmall-cell lung cancer (NSCLC); Or adenocarcinoma, can be for example colorectal carcinoma, cancer of pancreas or adenocarcinoma metastatic.Also comprise hematology's cancer.

Chimeric, humanized or people's antibody is in this paper scope, as the antibody of the cytotoxicity (ADCC) of mediate antibody dependent cell mediation.

In a preferred embodiment, described Anti-DR5 antibody comprises Apomab 7.3 or Apomab8.3, or its fragment.

Described antibody can be dimeric form and/or for example with the crosslinked form in anti-human IgG Fc district.

In other embodiments, described Anti-DR5 antibody herein and epitope tag sequence merge.

On the other hand, the present invention pays close attention to the chimeric molecule that the Anti-DR5 antibody comprise herein or antibody fragment and allogeneic amino acid sequence merge mutually, and wherein said allogeneic amino acid sequence can comprise for example immunoglobulin sequences, such as anti-human IgG Fc district.

Another aspect, the present invention pays close attention to the nucleic acid molecules of the separation of Anti-DR5 antibody herein of coding or antibody fragment, the carrier that comprises this type of nucleic acid molecules, the host cell that comprises this type of nucleic acid molecules, and produce antibody herein and the method for antibody fragment.

The invention still further relates to compositions, it comprises above defined Anti-DR5 antibody, and carrier.

Described carrier can be the pharmacopedics acceptable carrier, and described compositions also can comprise other anticarcinogen and/or other Anti-DR5 antibody.

Another aspect, the present invention pays close attention to apoptosis-induced method, comprises and makes mammalian cancer cells be exposed to above defined Anti-DR5 antibody.

Another aspect, the present invention pays close attention to the method for the treatment of cancer, comprises the above defined Anti-DR5 antibody of mammalian subject being used to effective dose.

In all respects, described experimenter can be human patients, and described cancer can be any cancer, comprises above listed cancer.

Aspect another one, the present invention pays close attention to goods, comprises container and is contained in the compositions in described container, and wherein said compositions comprises Anti-DR5 antibody of the present invention.Described goods also can be included in external or use in vivo the description of Anti-DR5 antibody.In a preferred embodiment, the treatment of the relevant cancer of described description.

The accompanying drawing summary

Fig. 1 has shown the nucleotide sequence (SEQ ID NO:2) of people's Apo2L/TRAIL cDNA and derivative aminoacid sequence (SEQ ID NO:1) thereof." N " that the 447th nucleotide (in SEQ ID NO:2) is located is for meaning that this nucleotide base can be " T " or " G ".

Fig. 2 A-2C has shown the nucleotide sequence (SEQ ID NO:4) of total length people DR4 receptor cdna and derivative aminoacid sequence (SEQ DD NO:3) thereof.The nucleotide sequence of people DR4 receptor and aminoacid sequence also are reported in Pan et al., Science, 276:111 (1997).

Fig. 3 A-3C has shown 411 amino acid whose sequences (SEQ ID NO:5) and the coding nucleotide sequence (SEQ ID NO:6) of people DR5 receptor, is disclosed in the WO98/51793 published on November 19th, 1998.

Fig. 4 A-4C has shown 440 amino acid whose sequences (SEQ ID NO:7) and the coding nucleotide sequence (SEQ ID NO:8) of people DR5, also is disclosed in the WO98/35986 published on August 20th, 1998.

Fig. 5 has shown the nucleotide sequence (SEQ ID NO:9) of strand Anti-DR5 antibody 16E2 (16E2 scFv).

Fig. 6 has shown the aminoacid sequence (SEQ ID NO:10) of strand Anti-DR5 antibody 16E2 (16E2 scFv), has wherein shown signal sequence and heavy chain and light chain CDR.

Fig. 7 has shown the aminoacid sequence (SEQ ID NO:11) of total length 16E2 heavy chain of antibody.

Fig. 8 has shown the nucleotide sequence (SEQ ID NO:12) of total length 16E2 heavy chain of antibody.

Fig. 9 has shown the aminoacid sequence (SEQ ID NO:13) of total length 16E2 light chain of antibody.

Figure 10 has shown the nucleotide sequence (SEQ ID NO:14) of total length 16E2 light chain of antibody.

Figure 11 A and B have shown the sequence (SEQID NO:15,5391bp) of the plasmid pDR1 for expressing light chain immunoglobulin.PDR1 comprises the irrelevant antibody of coding, the light chain of Humanized CD 3-resisting antibody, sequence (Shalaby et al., J.Exp.Med.175:217-225 (1992)), its initial sum termination codon indicates with runic and underscore.

Figure 12 A and B have shown the sequence (SEQID NO:16) of the plasmid pDR2 for expressing heavy chain immunoglobulin.PDR2 comprises the irrelevant antibody of coding, the heavy chain of Humanized CD 3-resisting antibody, sequence (Shalaby et al., supra), its initial sum termination codon indicates with runic and underscore.

Figure 13 has shown Apomab 7.3 heavy chain nucleotide sequences (SEQ ID NO:17).

Figure 14 has shown Apomab 7.3 heavy chain amino acid sequence (SEQ ID NO:18).

Figure 15 has shown Apomab 7.3 light chain nucleotide sequences (SEQ ID NO:19).

Figure 16 has shown Apomab 7.3 light-chain amino acid sequences (SEQ ID NO:20).

Figure 17 A and B have shown the heavy chain contrast of 16E2 and Apomab 7.3.

Figure 18 has shown the light chain contrast of 16E2 and Apomab 7.3.

Figure 19 is the homology model of Anti-DR5 antibody heavy chain.

Figure 20 is the homology model of Anti-DR5 antibody light chain.

Figure 21 has shown with total length 16E2 (pattern 1) antibody and has compared, the active anticancer in intraperitoneal (IP) single dose Apomab5.3,6.3 and 8.3 xenotransplantation of the Colo205 at human colon carcinoma nude mouse models.

Figure 22 has shown with total length 16E2 (pattern 1) and has compared, the active anticancer in IP single dose Apomabs 5.2,6.2,5.3,7.2 and 7.3 xenotransplantation of the Colo205 at human colon carcinoma nude mouse models.

Figure 23 has shown with total length 16E2 (pattern 1) and has compared, the active anticancer in IP single dose Apomab 5.2,7.3 and 8.3 xenotransplantation of the Colo205 at human colon carcinoma nude mouse models.

Figure 24 has shown with Apomab 7.3 and has compared, the active anticancer in IP single dose Apomab 23.3 and 25.3 xenotransplantation of the Colo205 at human colon carcinoma nude mouse models.

Figure 25 has shown the active anticancer of Apomab 7.3 in the Colo of human colon carcinoma 205 xenotransplantation nude mouse models than instantaneous cell line derived from stable cell lines.

Figure 26 has shown the active anticancer of Apomab 7.3 in the HCT15 of pulmonary carcinoma heteroplastic transplantation model independent and associating CPT-11.

Figure 27 has shown the active anticancer of Apomab 7.3 in the LS of people's sarcoma 180 heteroplastic transplantation models independent and associating CPT-11.

Figure 28 has shown independent and associating RITUXAN

(rituximab) active anticancer of Apomab 7.3 in the BJAB of non_hodgkin lymphoma xenotransplantation CB17 ICR SCID mouse model.

Figure 29 has shown the active anticancer of Apomab 7.3 in the BxPC3 of human pancreas's adenocarcinoma xenotransplantation nude mouse model independent and the associating gemcitabine.

Figure 30 has shown the active anticancer of Apomab 7.3 independent and associating carboplatin and taxol (taxol) at the H460 of people's pulmonary carcinoma heteroplastic transplantation model.

Figure 31 has shown Apomab 7.3 active anticancer in the H2122 of people's pulmonary carcinoma heteroplastic transplantation model independent and associating carboplatin and taxol.

Figure 32 has shown the dose-response curve of Apomab 7.3 in the H2122 of people's pulmonary carcinoma heteroplastic transplantation model.

Figure 33 has shown with Apomab 7.3 and has compared, the active anticancer in Apomab 23.3 and 25.3 205 heteroplastic transplantation models of the Colo at human colon carcinoma.

Figure 34 has shown independent Apo2L.0, independent Apomab 7.3 and multiple combination intermediate value tumor growth and the Kaplan-Meier figure to the 205 human colon carcinoma xenografts of Colo in nude mice.

Figure 35 and 36 has shown independent Apo2L.0, independent Apomab 7.3 and multiple combination intermediate value tumor growth and the Kaplan-Meier figure to SKMES-1 Non-small cell lung carcinoma (NSCLC) cell in the nude mouse heteroplastic transplantation model.

Figure 37 has shown that independent Apo2L.0, independent Apomab 7.3 and intermediate value tumor growth and the Kaplan-Meier of multiple combination in people Colo205 colon cancer heteroplastic transplantation model scheme.

The detailed description of preferred embodiment

I. definition

Term " Apo2L/TRAIL ", " Apo-2L ", " Apo2L ", " Apo2L/TRAIL/TRAIL " and " TRAIL " are used interchangeably in this article, refer to the peptide sequence that comprises amino acid residue 114-281 (containing), residue 95-281 (containing), residue 92-281 (containing), residue 91-281 (containing), residue 41-281 (containing), residue 39-281 (containing), residue 15-281 (containing) or the residue 1-281 (containing) of aminoacid sequence shown in Fig. 1 (SEQ ID NO:1), and the biological active fragment of above-mentioned sequence, deletion, insertion and/or alternative variations.In one embodiment, the residue 114-281 that peptide sequence comprises Fig. 1 (SEQ ID NO:1).Optional, residue 92-281 or residue 91-281 that peptide sequence comprises Fig. 1 (SEQ ID NO:1).The Apo-2L polypeptide can be by natural nucleotide sequential coding shown in Fig. 1 (SEQ ID NO:2).Optional, coding residue Pro119 (Fig. 1; SEQ ID NO:2) codon can be " CCT " or " CCG ".Optional is, described fragment or variant have biologic activity, and there is the amino acid sequence identity at least about 80% with arbitrary above-mentioned sequence, or at least about 90% sequence homogeneity, or at least 95%, 96%, 97%, 98% or 99% sequence homogeneity.The alternative variations of Apo2L/TRAIL is contained in this definition, and wherein its at least one natural amino acid substitutes with another kind of aminoacid such as alanine residue.The native sequences Apo2L/TRAIL separated from the Apo2L/TRAIL source or prepare by restructuring and/or synthetic method is also contained in this definition.Apo2L/TRAIL of the present invention comprise disclosed WO02/09755 of disclosed WO01/00832 of disclosed WO99/36535 of disclosed WO97/25428 of disclosed WO97/01633 on January 16th, 1997, on July 17th, 1997, on July 22nd, 1999, January 4 calendar year 2001, on February 7th, 2002, December in 2000 disclosed WO00/75191 on the 14th and on February 29th, 2000 bulletin United States Patent (USP) the 6th, the polypeptide that is called Apo2L/TRAIL or TRAIL disclosed in 030, No. 945.These terms are generally used for referring to the various forms of Apo2L/TRAIL, comprise monomer, dimer, trimer, six aggressiveness or the higher oligomer form of polypeptide.Except as otherwise noted, all amino acid residue numberings of mentioning in the Apo-2L sequence adopt the numbering according to Fig. 1 (SEQ ID NO:1).

" Apo2L/TRAIL receptor " comprises that this area is called the receptor of " DR4 " and " DR5 ", and its polynucleotide and peptide sequence are shown in Fig. 2 A-2C (SEQ ID NO:4 and 3) and 3A-3C (SEQID NO:6 and 5).The people such as Pan have described TNF receptor family member (Pan et al., Science, the 276:111-113 (1997) that is called " DR4 "; WO98/32856, be disclosed on July 30th, 1998; WO99/37684, be disclosed on July 29th, 1999; WO00/73349, be disclosed in December in 2000 7; US6,433,147, be issued on August 13rd, 2002; US6,461,823, be issued on October 8th, 2002; US6,342,383, be issued on January 29th, 2002).The people such as Sheridan (Scierce, 277:818-821 (1997)) and the people (Science such as Pan, 277:815-818 (1997)) the another kind of receptor of having described Apo2L/TRAIL (also can, referring to WO98/51793, be disclosed on November 19th, 1998; WO98/41629, be disclosed in JIUYUE in 1998 24).This receptor is called DR5, and (this receptor also can be called Apo-2, TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER; Screaton et al., Curr.Biol., 7:693-696 (1997); Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO98/35986, be disclosed on August 20th, 1998; EP870,827, be disclosed on October 14th, 1998; WO98/46643, be disclosed on October 22nd, 1998; WO99/02653, be disclosed on January 21st, 1999; WO99/09165, be disclosed on February 25th, 1999; WO99/11791, be disclosed on March 11st, 1999; US2002/0072091, be disclosed on August 13rd, 2002; US2002/0098550, be disclosed in calendar year 2001 December 7 days; US6,313,269, be issued to calendar year 2001 December 6 days; US2001/0010924, be disclosed in August 2 calendar year 2001; US2003/01255540, be disclosed on July 3rd, 2003; US2002/0160446, be disclosed on October 31st, 2002; US2002/0048785, be disclosed on April 25th, 2002; US6,569,642, be issued on May 27th, 2003; US6,072,047, be issued on June 6th, 2000; US6,642,358, be issued on November 4th, 2003).As mentioned above, other receptor of Apo-2L comprises that DcR1, DcR2 and OPG (referring to Sheridan et al., see above; Marsters et al., see above; Simonet et al., see above).Native sequences receptor and receptor variant contained in term " Apo-2L receptor " for this paper the time.These terms are encompassed in multiple mammal and comprise the Apo-2L receptor of expressing in the people.The Apo-2L receptor can be endogenous expression, as organized natural generation in pedigree in various human, or can express by restructuring or synthetic method." native sequences Apo-2L receptor " comprises and has the polypeptide of same acid sequence derived from natural Apo-2L receptor.Therefore, native sequences Apo-2L receptor can have the aminoacid sequence that comprises people's the natural Apo-2L of existence receptor from any mammal.This type of native sequences Apo-2L receptor can separate from nature, or can be by restructuring or synthesizing mean production.Receptor (for example containing for example soluble form of extracellular domain sequence), naturally occurring variant form (for example alternative splicing form) and the naturally occurring allele variant of naturally occurring truncate or secreted form clearly contained in term " native sequences Apo-2L receptor ".The receptor variant can comprise the fragment of native sequences Apo-2L receptor or delete mutant.Fig. 3 A-3C has shown 411 amino acid whose people DR5 sequences that disclose in disclosed WO98/51793 on November 19th, 1998.A kind of splice variant of transcribing of people DR5 is known in this area.440 amino acid whose people DR5 sequences shown in this DR5 splice variant code pattern 4A-4C, and nucleotide sequence (SEQID NO:7 and 8), as disclosed on August 20th, 1998 disclosed WO98/35986.

" death receptor antibody " is often referred to the antibody of the receptor that comprises the death domain that can send apoptotic signal for tumor necrosis factor receptor super family for this paper the time, and this antibody-like comprises DR5 antibody and DR4 antibody.

" DR5 receptor antibody ", " DR5 antibody " or " Anti-DR5 antibody " are used with broad sense, refer in conjunction with the DR5 receptor of at least one form or the antibody of its extracellular domain.Optional, DR5 antibody merges or is connected with heterologous sequence or molecule.Preferably, heterologous sequence is allowed or is helped antibody to form more high-grade or oligomeric complex.Optional, DR5 antibodies DR5 receptor but not with any other Apo-2L receptor (for example DR4, DcR1 or DcR2) in conjunction with or cross reaction occurs.Optional, this antibody is the agonist of DR5 signal activity.Term " Anti-DR5 antibody " and grammer equivalent thereof are clearly contained the antibody described in embodiment, include but not limited to " Apomab " antibody listed in table 11 and 12, for example Apomab 1.1,2.1,3.1,4.1,5.1,6.1,7.1,8.1,9.1,1.2,2.2,3.2,4.2,5.2,6.2,7.2,8.2,9.2,1.3,2.3,3.3,4.3,5.3,6.3,7.3,8.3 and 9.3, preferably Apomab 7.3.

Optional, according to the measurement of BIAcore binding assay, DR5 antibody of the present invention about 0.1nM in the concentration range of about 20mM in conjunction with the DR5 receptor.Optional, according to the measurement of BIAcore binding assay, DR5 antibody of the present invention presents the Ic50 value of about 0.6nM to about 18mM.

" DR4 receptor antibody ", " DR4 antibody " or " anti-DR4 antibody " are used with broad sense, refer in conjunction with the DR4 receptor of at least one form or the antibody of its extracellular domain.Optional, DR4 antibody merges or is connected with heterologous sequence or molecule.Preferably, heterologous sequence allows or helps antibody to form more high-grade or oligomeric complex.Optional, DR4 antibodies DR4 receptor but not with any other Apo-2L receptor (for example DR5, DcR1 or DcR2) in conjunction with or cross reaction occurs.Optional, this antibody is the agonist of DR4 signal activity.

Optional, according to the measurement of BIAcore binding assay, DR4 antibody about 0.1nM in the concentration range of about 20mM in conjunction with the DR4 receptor.Optional, according to the measurement of BIAcore binding assay, DR4 antibody of the present invention presents the Ic50 value of about 0.6nM to about 18mM.

Term " agonist " is used with broad sense, is included in external, original position or partially or completely strengthens in vivo, stimulates or activate any molecule of one or more biologic activity of Apo2L/TRAIL, DR4 or DR5.Apo2L/TRAIL comprises in conjunction with the example of this type of biologic activity of DR4 or DR5 those that further report in apoptosis and document.Agonist directly or indirectly mode is brought into play function use.For example, thus agonist can be in vitro, original position or directly in conjunction with DR4 or DR5, cause the result performance function of receptor activation or signal transduction due to it in vivo and partially or completely strengthen, stimulate or activate one or more biologic activity of DR4 or DR5.Agonist can also be in vitro, original position or in vivo owing to for example stimulating another kind of effector molecule then to cause that the result of DR4 or DR5 activation or signal transduction indirectly brings into play function and partially or completely strengthen, stimulate or activate one or more biologic activity of DR4 or DR5.Imagine agonist and can be used as the reinforcing agent molecule that indirect performance function strengthens or improve DR4 or DR5 activation or activity.For example, agonist can strengthen the activity of endogenous Apo-2L in mammal.This can be by for example compound (pre-complexing) DR4 or DR5 or realize by the complex (such as stablizing the natural complex formed between Apo-2L and DR4 or DR5) of stablizing each part and DR4 or DR5 receptor in advance.

" ectodomain " or " ECD " assignment body or receptor are substantially free of the form in membrane spaning domain and cytoplasmic structure territory.Usually, soluble E CD will have this type of membrane spaning domain and the cytoplasmic structure territory that is less than 1%, preferably will have this type of domain that is less than 0.5%.

Term " Epitope tag " refers to comprise protein such as Apo2L/TRAIL or DR5 receptor or its part or the chimeric polyeptides that merges in conjunction with the antibody of this type of part or receptor and itself and " label polypeptide " for this paper the time.The label polypeptide has enough residues can prepare the antibody for it so that epi-position to be provided, but enough short activity that makes it not disturb part or receptor.The label polypeptide preferably or fairly individual, make its antibody basically not with other epi-position generation cross reaction.Suitable label polypeptide has at least 6 amino acid residues usually, usually approximately 8 and approximately between 50 amino acid residues (preferably approximately 10 and approximately between 20 residues).

" separation ", when describing range protein disclosed herein, mean to identify and with/the protein that separates and/or reclaim by a kind of composition of its natural surroundings.The contaminative composition of its natural surroundings refers to usually to disturb the diagnosis of this protein or the material of therapeutic use, can comprise the solute of enzyme, hormone and other oroteins character or nonprotein character.In preferred embodiments, protein purification to (1) is enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (2) reach homogeneity according to using Coomassie blue or the irreducibility of preferably silver dyeing or the SDS-PAGE under reductive condition.Since at least one composition of Apo2L/TRAIL natural surroundings can not exist, the protein separated so comprises the original position protein in reconstitution cell.Yet usually prepared by least one purification step by the protein of separation.

" percentage ratio (%) amino acid sequence identity " about the sequence identified herein is defined as the contrast sequence and introduces where necessary breach with after obtaining largest percentage sequence homogeneity, and not by any conservative substituting while being considered as sequence homogeneity a part of, the percentage rate of the amino acid residue identical with amino acid residue in compared part, receptor or antibody sequence in candidate sequence.Various ways for the contrast of measuring percentage ratio amino acid sequence identity purpose in can the art technology scope carries out.Those skilled in the art can determine for measuring the suitable parameter of contrast, comprise that assessment obtains the required algorithm of maximum contrast to compared full length sequence.For the purposes of the present invention, percentage ratio amino acid sequence identity value can be used relatively computer program ALIGN-2 acquisition of sequence, it is write by Genentech company, its source code is submitted to (the US Copyright Office of U.S. Copyright Bureau together with customer documentation, Washington D.C., 20559), and with U.S. copyright registration TXU510087 register.The public can pass through Genentech company (South San Francisco, CA) and obtain the ALIGN-2 program.The all sequences comparative parameter is by ALIGN-2 program setting and constant.Then calculate the percentage ratio amino acid sequence identity with respect to longer sequence.Therefore, even shorter sequence is completely contained in longer sequence, sequence homogeneity also will be less than 100%.

Term " control sequence " refers to express in the specific host organism the necessary DNA sequence of coded sequence be operatively connected.For example, be suitable for procaryotic control sequence and comprise promoter, optional operator sequence and ribosome binding site.The known genuine nucleus utilizes promoter, polyadenylation signal and enhancer.

If one section nucleic acid and another section nucleotide sequence are in functional mutual relation, it is " being operatively connected ".For example, if presequence (presequence) or the DNA that secretes leading (secretory leader) are expressed as the front protein (preprotein) that participates in the polypeptide secretion, the DNA of it and polypeptide is operatively connected; If promoter or enhancer affect transcribing of coded sequence, it and this sequence are operatively connected; Perhaps, if the position of ribosome binding site promotes translation, it and coded sequence are operatively connected.Usually, " being operatively connected " means that connected DNA sequence is adjacent, and in the leading situation of secretion, means adjacent and in read state.Yet enhancer needn't be adjacent.Connection can be by realizing in the coupled reaction at restriction site place easily.If there is no this type of site, use synthetic oligonucleotide adapter or joint according to conventional practice so.

The polyhydroxy-alcohol compound made a general reference in term " polyhydric alcohol (polyol) " for this paper the time.Polyhydric alcohol can be any water-soluble poly (alkylene oxide) polymer for example, and can have linear chain or branched chain.Preferred polyhydric alcohol comprises the polyhydric alcohol that those replace such as the alkyl with 1 to 4 carbon with chemical group in one or more hydroxy position.Be typically, polyhydric alcohol is poly-(aklylene glycol), preferably PEG (PEG).Yet, those skilled in the art recognize that, other polyhydric alcohol can utilize the coupling technology of describing about PEG to be adopted herein such as poly-(propylene glycol) and polyethylene-polypropylene glycol copolymer.Polyhydric alcohol comprises those types well-known in the art and those types that can openly obtain, such as obtaining from commercial source, such as Nektar

corporation.

Term " coupling " (conjugate) defines use according to it in this article the most widely, and finger closes or connects together.If molecule works or operates when being bonded together, they are " couplings ".

" stringency " of hybridization can be easy to determine by those of ordinary skills, and usually calculate by rule of thumb according to probe length, wash temperature and salinity.Generally speaking, the temperature that longer probe is had relatively high expectations is correctly to anneal, and shorter probe needs lower temperature.Hybridization depends on the ability that time variation DNA anneals again in complementary strand is present in lower than the environment of its melting temperature usually.But the expectation homogeneity degree between probe and hybridization sequences is higher, and spendable relative temperature is also higher.Result is infer that higher relative temperature will trend towards making reaction condition more strict, and lower temperature to be just not strict yet.About other details and the explanation of hybridization stringency, referring to people such as Ausubel, " Current Protocols in Molecular Biology ", Wiley Interscience Publishers, 1995.

" stringent condition " or " high stringency ", as defined herein, identify as follows: (1) adopts low ionic strength and high temperature to be cleaned, and 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulphate for example, in 50 ℃; (2) adopt denaturant in crossover process, such as Methanamide, 50% (v/v) Methanamide and 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer pH6.5 and 750mM sodium chloride for example, the 75mM sodium citrate, in 42 ℃; Or (3) adopt 50% Methanamide, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% tetrasodium pyrophosphate, 5x DenhardtShi solution, the salmon sperm DNA of supersound process (50 μ g/ml), 0.1%SDS, with 10% dextran sulfate, in 42 ℃, and clean in 0.2x SSC (sodium chloride/sodium citrate) and 50% Methanamide in 42 ℃, then in 55 ℃, containing in the 0.1x SSC of EDTA, carrying out high stringency cleaning.

" medium stringent condition " can be as people such as Sambrook, " Molecular Cloning:ALaboratory Manual ", New York, Cold Spring Harbor Press, identify described in 1989, comprise and for example using, than more undemanding cleaning solution mentioned above and hybridization conditions (temperature, ionic strength and %SDS).An example of medium stringent condition is to contain 20% Methanamide in 37 ℃, 5x SSC (150mM NaCl, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x DenhardtShi solution, 10% dextran sulfate, and be incubated overnight in the solution of the salmon sperm DNA of 20mg/ml degeneration shearing, then in about 37-50 ℃ of cleaning filter membranes in 1x SSC.The technical staff will recognize and adjust how where necessary temperature, ionic strength etc. to adapt to such as factors such as probe length.

Term " aminoacid " refers to all naturally occurring L-a-amino acids.This definition intention comprises nor-leucine, ornithine and homocysteine.Aminoacid indicates and differentiates by single-letter or trigram:

Asp D aspartic acid Ile I isoleucine

Thr T threonine Leu L leucine

Ser S serine Tyr Y tyrosine

Glu E glutamic acid Phe F phenylalanine

Pro P proline His H histidine

Gly G glycine Lys K lysine

Ala A alanine Arg R arginine

Cys C cysteine Trp W tryptophan

Val V valine Gln Q glutamine

Met M methionine Asn N agedoite

In the accompanying drawings, can adopt some other single-letter or trigram to indicate to point to and identify two or more aminoacid or the nucleotide of given position in sequence.

Term " antibody " is used with broad sense, clearly covers single anti-DR5 monoclonal antibody (comprising excitability, Antagonism and neutrality or blocking antibody) and has the specific Anti-DR5 antibody compositions of multi-epitope." antibody " comprises complete immunoglobulin or antibody molecule, polyclonal antibody, multi-specificity antibody (bi-specific antibody for example formed by least two kinds of complete antibody), reaches immunoglobulin fragment (such as Fab, F (ab ') for this paper the time ₂or Fv), as long as they show the described herein excitability how to expect or Antagonism characteristic.

Antibody is typically protein or the polypeptide shown the binding specificity of specific antigen.The different tetramer glycoprotein that natural antibody normally consists of two identical light chains (L) and two identical heavy chains (H).Typically, every light chain is connected with heavy chain by a covalent disulfide bonds, and the disulfide bond number changes to some extent between the heavy chain of different Immunoglobulin Isotypes.The intrachain disulfide bond that every heavy chain and light chain also have the interval rule.Every heavy chain has a variable region (VH) at one section, is then a plurality of constant regions.Every light chain at one end has a variable region (V _l), the other end is a constant region.Constant region of light chain and heavy chain the first constant region are arranged together, and variable region of light chain and variable region of heavy chain are arranged together.Think that specific amino acid residue forms interface (Chothia et al., J.MoI.Biol., 186:651-663 (1985) between light chain and variable region of heavy chain; Novotny and Haber, Proc.Natl.Acad.Sci.USA, 82:4592-4596 (1985)).According to its constant region aminoacid sequence, can be included into a kind of in two kinds of completely different types from the light chain of antibody of any invertebrate species, be called card handkerchief (κ) and lambda (λ).According to its CH aminoacid sequence, immunoglobulin can be included into different classifications.Five large immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM are arranged, and wherein some can be further divided into subclass (isotype), for example IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2.CH corresponding to the inhomogeneity immunoglobulin is called respectively α, δ, ε, γ and μ.

The part that " antibody fragment " comprises complete antibody, the normally combination of the antigen of complete antibody or variable region.The example of antibody fragment comprises Fab, Fab ', F (ab ') ₂with the Fv fragment; Double antibody; The single-chain antibody molecule; And the multi-specificity antibody formed by antibody fragment.

Term " variable " in this article for some part of describing variable region between antibody sequence variant and for every kind of specific antibodies combination and the specificity to its specific antigen.Yet variability is not the whole variable region that is uniformly distributed in antibody usually.It typically concentrates in light chain and variable region of heavy chain three sections that are called complementary determining region (CDR) or hypervariable region.In variable region more the part of high conservative be called framework region (FR).Each self-contained four FR of the variable region of natural heavy chain and light chain, they take the beta-pleated sheet conformation mostly, and three CDR that form a beta-pleated sheet structure part by the formation loop connecting and in some situation connect.What the CDR in every chain approached by FR very much keeps together, and with the formation of the antigen binding site of facilitating antibody together with the CDR of another chain (referring to Kabat, E.A. wait the people, " Sequences of Proteins of Immunological Interest ", National Institutes ofHealth, Bethesda, MD, 1987).Constant region is not participated in the combination of antibody and antigen directly, but shows multiple effector function, such as the participation of antibody in antibody dependent cellular cytotoxicity.

Term " monoclonal antibody " refers to that for this paper the time each antibody that forms colony is identical from a group antibody that the antibody of homogeneity obtains basically, except may be with indivisible possible natural the existence sudden change existed.Monoclonal antibody is high degree of specificity, for single antigenic site.In addition, from routine (polyclone) the antibody preparations difference typically comprised for the different antibodies of different determinants (epi-position), every kind of monoclonal antibody is for the single determinant on antigen.

Monoclonal antibody comprise in this article variable region (comprising hypervariable region) by Anti-DR5 antibody and constant region (for example " humanization " antibody) or light chain and antibody or chimeric, the heterozygosis that produces from the montage of chain of species and a chain from another species with the antibody of recombinating, or with the fusions of heterologous protein, no matter the class of On the Origin of Species or immunoglobulin or subclass ownership, and antibody fragment (for example Fab, F (ab ') ₂and Fv), as long as they show biologic activity or the characteristic of expectation.Referring to for example United States Patent (USP) the 4th, 816, No. 567 and Mage et al., in " Monoclonal Antibody Production Techniques and Applications ", pp.79-97, Marcel Defcker, Inc.:New York, 1987.

So, modifier " monoclonal " indication antibody, from the feature that the antibody population of homogeneity obtains basically, should not be construed as and requires to generate antibody by any ad hoc approach.For example, the monoclonal antibody that to use according to the present invention can be passed through at first by Kohler and Milstein, prepared by the hybridoma method of Nature256:495 (1975) record, or can pass through such as United States Patent (USP) the 4th, prepared by the recombinant DNA method of describing in 816, No. 567." monoclonal antibody " also can be from using for example McCafferty et al., Nature, and the phage antibody library that in 348:552-554 (1990), the technology of record produces separates.

" humanization " form of inhuman (for example Mus) antibody refers to that bottom line comprises specific gomphosis immunoglobulin, immunoglobulin chain or its fragment derived from the sequence of non-human immunoglobulin (such as Fv, Fab, Fab ', F (ab ') ₂or other antigen zygote sequence of antibody).Largely, humanized antibody refers to the immunoglobulin that complementary determining region (CDR) residue in human normal immunoglobulin's (receptor antibody) is replaced with the CDR residue of inhuman species (donor antibody) such as mice, rat or rabbit with expectation specificity, affinity and ability.In some situation, human normal immunoglobulin's Fv framework region (FR) residue is replaced with corresponding inhuman residue.In addition, humanized antibody can be included in the residue all do not found in receptor antibody or input CDR or Frame sequence.Carrying out these modifications is in order further to improve and optimize the performance of antibody.Generally speaking, humanized antibody will comprise at least one, common two whole following variable regions basically, wherein whole or basically whole CDR corresponding to the hypermutation ring of non-human immunoglobulin, and whole or basically whole FR be the FR of human normal immunoglobulin's consensus sequence.Humanized antibody preferably also will comprise at least part of constant region for immunoglobulin (Fc), normally human normal immunoglobulin's constant region.

" people's antibody " refers to have the aminoacid sequence corresponding with the aminoacid sequence of the antibody generated by the people and/or uses the antibody generated for any technology that generates people's antibody known in the art or disclosed herein.This definition of people's antibody comprises the antibody that comprises at least one people's heavy chain polypeptide or at least one people's light chain polypeptide, for example comprises the antibody of Mus light chain and people's heavy chain polypeptide.People's antibody can generate by multiple technologies known in the art.In one embodiment, people's antibody is selected from phage library, and this phage library is expressed people's antibody (Vaughan et al., Nature Biotechnology, 14:309-314 (1996); Sheets et al., PNAS (USA) 95:6157-6162 (1998); Hoogenboom and Winter, J.Mol.Biol..227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991)).People's antibody also can by the human immunoglobulin gene is organized import endogenous immunoglobulin gene partially or completely the transgenic animal of deactivation for example mice generate.When under attack, observe people's antibody and generate, it in all respects in human body, see extremely similar, comprise gene rearrangement, assembling and antibody complete or collected works.This method is recorded in for example United States Patent (USP) 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and following scientific publications: Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-13 (1994); Fishwild et al., Nature Biotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern.Rev.Immunol., 13:65-93 (1995).Perhaps, people's antibody can prepare (this type of bone-marrow-derived lymphocyte can reclaim from individuality, or immunity in vitro) by the immortalization generated for the human B lymphocyte of the antibody of target antigen.Referring to for example Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); Boemer et al., J.Immunol., 147 (1): 86-95 (1991); United States Patent (USP) the 5th, 750, No. 373.

Term " Fc district " is for defining the C-end region of heavy chain immunoglobulin, and it can be by producing with the papain digestion complete antibody.The Fc district can be native sequences Fc district or variant Fc regions.Although the border in heavy chain immunoglobulin Fc district can change to some extent, yet human IgG heavy chain Fc district is normally defined near the Cys226 position or near the amino acid residue Pro230 position to the section (using the al. according to Kabat et herein, the numbering system of supra) of the carboxyl terminal in Fc district.The Fc district of immunoglobulin comprises two constant regions usually, and CH2 domain and CH3 domain, optionally comprise the CH4 domain.

" Fc district chain " refers to one of two polypeptide chains in Fc district in this article.

" the CH2 domain " in human IgG Fc district (also referred to as " C γ 2 " domain) extends to approximately the 340th amino acids residue from about the 231st amino acids residue usually.The unique distinction of CH2 domain is that it closely not match with another domain.But the carbohydrate chain of the branch that has two N-to connect is between two CH2 domains of complete natural IgG molecule.Infer that carbohydrate may provide substituting of domain-domain pairing and contribute to stablize the CH2 domain.Burton，Molec.Immunol.22：161-206(1985)。The CH2 domain can be native sequences CH2 domain or variation CH2 domain in this article.

One section residue that " CH3 domain " comprises CH2 domain C-end in the Fc district (from approximately the 341st amino acids residue of IgG to about the 447th amino acids residue).The CH3 district can be that (" protuberance " that for example in one bar chain, has an introducing (protroberance) has " cavity " CH3 domain (cavity) of corresponding introducing in its another chain for native sequences CH3 domain or variation CH3 domain in this article; Referring to United States Patent (USP) 5,821,333).This type of variation CH3 domain can be used for generating polyspecific described herein (for example bispecific) antibody.

" hinge region " is normally defined the section (Burton, Molec.Immunol.22:161-206 (1985)) to about Pro230 from human IgG1's approximately Glu216 or about Cys226.The hinge region of other IgG isotype can be by being placed in first and the cysteine residues that last forms S-S key between heavy chain same position and contrasting with the IgG1 sequence.Hinge region can be native sequences hinge region or variation hinge region in this article.Two polypeptide chains of variation hinge region every polypeptide chain usually retain at least one cysteine residues, make two polypeptide chains of variation hinge region to form disulfide bond between two chains.Preferred hinge region is native sequences people hinge region, for example native sequences human IgG1 hinge region herein.

" functional Fc district " has at least one " effector function " in native sequences Fc district.Exemplary " effector function " comprises the C1q combination; CDC (CDC); The Fc receptors bind; The cytotoxicity (ADCC) of antibody dependent cellular mediation; Phagocytosis; Cell surface receptor (B-cell receptor for example; BCR) lower; Deng.This type of effector function requires the Fc district for example, to combine with binding structural domain (antibody variable region) usually, and the many measure method for assessment of this type of antibody mediated effect thing function that can know with this area is assessed.

The identical aminoacid sequence of aminoacid sequence that " native sequences Fc district " comprises the Fc district found with occurring in nature." variant Fc regions " comprises the aminoacid sequence different from the aminoacid sequence in native sequences Fc district because at least one place is amino acid modified.Preferably, variant Fc regions is compared and is had at least one place amino acid replacement with native sequences Fc district or with the Fc district of parent's polypeptide, for example in native sequences Fc district or in the Fc district of parent's polypeptide approximately a place to about ten place's amino acid replacements, preferably approximately a place to about five place's amino acid replacements.Variant Fc regions in this article will be preferably and native sequences Fc district and/or have at least about 80% sequence homogeneity with the Fc district of parent's polypeptide, most preferably at least about 90% sequence homogeneity, more preferably at least about 95% sequence homogeneity.

" cytotoxicity of antibody dependent cellular mediation " and " ADCC " refer to by cell-mediated reaction, the non-specific cell toxic cell (for example NK cell (NK) cell, neutrophil cell and macrophage) of wherein expressing Fc receptor (FcR) is identified the antibody of combination on target cell, causes subsequently the target cell dissolving.The main cell of mediation ADCC, the NK cell, only express Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 has been summed up the FcR on the hematopoietic cell and has been expressed.For the ADCC activity of purpose of appraisals molecule, can carry out external ADCC algoscopy, such as United States Patent (USP) the 5th, 500,362 or 5,821, put down in writing in No. 337.The effector lymphocyte who can be used for this type of algoscopy comprises peripheral blood lymphocytes (PBMC) and NK cell (NK) cell.Perhaps/in addition, the ADCC activity of purpose of appraisals molecule in vivo, for example, in animal model, such as Clynes et al., disclosed in PNAS (USA) 95:652-656 (1998).

The leukocyte that " people effector lymphocyte " refers to express one or more FcR and exercise effector functions.Preferably, this cell is at least expressed Fc γ RIII and is exercised the ADCC effector functions.The example of the human leukocyte of mediation ADCC comprises PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC), NK cell (NK) cell, mononuclear cell, cytotoxic T cell and neutrophil cell, preferably PBMC and NK cell.The effector lymphocyte can separate from its natural origin, for example blood or PBMC, as described in this article.

Term " Fc receptor " and " FcR " are for describing the receptor in binding antibody Fc district.Preferred FcR is native sequences people FcR.In addition, preferred FcR is the FcR (γ receptor) in conjunction with IgG antibody, comprises the receptor of Fc γ RI, Fc γ RII and Fc γ RIII subclass, comprises allelic variant and the alternative splicing form of these receptors.Fc γ RII receptor comprises Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" inhibition receptor "), and they have similar aminoacid sequence, and difference mainly is its cytoplasmic structure territory.Activated receptor Fc γ RIIA comprises the activation motif (ITAM) of immunity receptor based on tyrosine in its cytoplasmic structure territory.Suppress receptor Fc γ RIIB and comprise the inhibition motif (ITIM) (referring to summary Da ё ron, Annu.Rev.Immunol.15:203-234 (1997)) of immunity receptor based on tyrosine in its cytoplasmic structure territory.The summary of FcR is referring to Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); De Haas et al., J.Lab.Clin.Med.126:330-41 (1995).Other FcR contained in this article in term " FcR ", comprises what will identify those futures.This term also comprises the neonate receptor, FcRn, and it is responsible for Maternal immunoglobulin G is transferred to fetus (Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.Immunol.24:249 (1994)).

" CDC " and " CDC " refers to dissolve the target thing when having complement.The complement activation approach is for example, in conjunction with the molecule (antibody) compound with closing associated antigen initial by complement system the first component (C1q).In order to assess complement activation, can carry out the CDC algoscopy, for example, as Gazzano-Santoro etal., in J.Immunol.Methods 202:163 (1996), put down in writing.

" affinity maturation " antibody refers to have in its one or more CDR and causes antibody to compare to the affinity of antigen the antibody that the place that improves to some extent or many places change with the parental antibody that there is no these changes.The antibody of preferred affinity maturation will have nanomole or the affinity to target antigen of picomole level even.The antibody of affinity maturation can generate by flow process known in the art.Marks et al., Bio/Technology10:779-783 (1992) has put down in writing by the affinity maturation of VH and the reorganization of VL domain.Following document has been put down in writing the random mutagenesis of CDR and/or framework residue: Barbas et al., Proc.Nat.Acad.Sci.USA91:3809-3813 (1994); Schier et al., Gene169:147-155 (1995); Yelton et al., J.Immunol.155:1994-2004 (1995); Jackson et al., J.Immunol.154 (7): 3310-9 (1995); Hawkins et al., J.Mol.Biol.226:889-896 (1992).

The antigenic specificity binding interactions occurred between the specific antigen that term " immunologic opsonin " refers to identify at antigen binding site and this antibody of antibody for for example the immunologic opsonin of the antibody " in conjunction with " time.

For the purposes of the present invention, " biologic activity " and " biologic activity of expectation " refers to have the ability in vivo or in vitro (ex vivo) regulates and controls DR5 activity or DR5 activation in the mammalian cell of at least one type, comprise for example apoptosis (or excitability or zest mode or Antagonism or barrier mode), in conjunction with Apo2L/TRAIL (TRAIL), or the activation of one or more molecules such as caspase 3, Caspase 8, Caspase 10 or FADD in regulation and control intracellular signal approach.For measuring the algoscopy of molecule activation in these type of born of the same parents, be known in the art, referring to for example Boldin et al., J.Biol.Chem., 270:7795-7798 (1995); Peter, Cell Death Differ., 7:759-760 (2000); Nagata, Cell, 88:355-365 (1998); Ashkenazi et al., Science, 281:1305-1308 (1999).

The molecule of can direct or indirect substance inducing, promote or strengthen DR5 biologic activity or activation is pointed to or described to term " agonist " and " excitability " for this paper the time.Optional is, " agonist DR5 antibody " refers to have the part with DR5, be called Apo2L/TRAIL (TRAIL), suitable activity, thereby perhaps can activate the antibody that the DR5 receptor causes one or more intracellular signal pathway activations, it can comprise the activation of caspase 3, Caspase 8, Caspase 10 or FADD.

Term " antagonist " and " Antagonism " point to or describe substance counteracting, reduction that can be direct or indirect or suppress the DR5 biologic activity or the molecule of DR5 activation for this paper the time.Optional, antagonist refers to that neutralization is derived from the molecule that DR5 activation or DR5 and its part such as Apo2L/TRAIL form the biologic activity of complex.

Term " apoptosis " and " apoptosis activity " are used with broad sense, the cell death that refers to orderly in mammal or controlled form, usually be accompanied by one or more characteristic cellular change, comprise that Cytoplasm is concentrated, forfeitures of plasma membrane microvillus, nucleus segmentation, chromosomal DNA degraded or mitochondrial function forfeiture.Can measure and measure this activity by for example cells survival amylograph, annexin V binding assay, PARP algoscopy, facs analysis or DNA electrophoresis, these are all known in the art.Optional, measure apoptosis activity by annexin V or PARP algoscopy.

Term " cancer ", " carcinous " and " malignant tumor " are pointed to or describe feature in mammal and be generally the not modulated physiology illness of Growth of Cells.The example of cancer includes but not limited to cancer, comprises adenocarcinoma, lymphoma, blastoma, melanoma, (nerve) glioma, sarcoma, myeloma (such as multiple myeloma) and leukemia.The more specifically example of this type of cancer comprises squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, the adenocarcinoma of lung, the squamous cell carcinoma of lung, human primary gastrointestinal cancers, He Jiejin lymphomas and non_hodgkin lymphoma, cancer of pancreas, glioblastoma, cervical cancer, (nerve) glioma, ovarian cancer, the cancer of liver (liver cancer) is such as hepatocarcinoma (hepatic carcinoma) and hepatoma (hepatoma), bladder cancer, breast carcinoma, colon cancer, colorectal carcinoma, endometrium or uterus carcinoma, salivary-gland carcinoma, the cancer of kidney (kidney cancer) such as renal cell carcinoma (renal cell carcinoma) and Wei Ermusishi tumor (Wilms ' tumor), basal cell carcinoma, melanoma, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, carcinoma of testis, esophageal carcinoma, and various types of heads and neck cancer.

Term " immune correlated disease " refers to be caused, mediate or otherwise facilitated by the immune system composition in mammal the disease of morbidity.Also comprise stimulating or intervening immunne response and disease progression is there is to the disease of improvement effect.This term comprises inflammatory diseases, infectious disease and the immunodeficiency of autoimmune disease, immune-mediated inflammatory diseases, nonimmune mediation.Can be according to the Immune interrelation of the present invention's treatment and the example of inflammatory diseases, wherein some is that immunity or T are cell-mediated, comprises systemic lupus erythematosus (sle), rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Si Yegelunshi (Sjogren) syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immunopancytopenia, paroxysmal nocturnal hemoglobinuria), AT (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Ge Leifusishi (Graves) disease, Hashimoto disease (Hashimoto) thyroiditis, the juvenile form lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis), the demyelinating disease of maincenter and peripheral nervous system is such as multiple sclerosis, idiopathic demyelination polyneuropathy or Ge-Ba Er Shi (Guillain-Barr é) syndrome, with chronic inflammatory demyelination polyneuropathy, liver-gallbladder disease is such as infectious hepatitis (A type, B-mode, the third type, the fourth type, penta type and other non-Hepadna Virus hepatitis), ACAH, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrosis pneumonopathy are such as inflammatory bowel (ulcerative colitis, Crow engler (Crohn) disease), gluten sensitive enteropathy, with the Er Shi of Hewlett-Packard (Whipple) disease, autoimmunity or immune-mediated dermatosis comprise bullous dermatosis, erythema multiforme and contact dermatitis, psoriasis, allergic disease is such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, the amynologic disease of lung is such as the eosinocyte pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplant relevant disease and comprise transplant rejection and graft versus host disease.Infectious disease comprises AIDS (HIV infection), A type, B-mode, the third type, fourth type and hepatitis E, antibacterial infection, fungal infection, protozoal infections and parasitic infection.

" autoimmune disease " used with broad sense, general significance in this article, refers in mammal because of mammalian subject the body fluid of himself structural constituent or disease or the illness that cellullar immunologic response destroys normal or health tissues.Example includes but not limited to lupus erythematosus, thyroiditis, rheumatoid arthritis, psoriasis, multiple sclerosis, Autoimmune Diabetes and inflammatory bowel (IBD).

" growth inhibitor " refers in vitro and/or cytostatic compound or compositions in vivo for this paper the time.So, growth inhibitor can be significantly to reduce the medicament of the cell percentage ratio in the S phase.The example of growth inhibitor comprises the advance medicament of (position beyond the S phase) of blocking-up cell cycle, such as inducing the medicament that G1 stagnates and the M phase stagnates.Classical M phase blocker comprises Changchun medicine class (vincas) (vincristine (vincristine) and vinblastine (vinblastine)), TAXOL , and Topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide) and bleomycin (bleomycin).Medicaments of those retardances G1 also overflow and enter the S phase and stagnate, for example DNA alkylating agent class such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), chlormethine (mechlorethamine), cisplatin (cisplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil) and ara-C.More information can be referring to " The Molecular Basis of Cancer ", Mendelsohn and Israel compile, the 1st chapter, be entitled as " Cell cycle regulation, oncogenes; and antineoplastic drugs ", the people such as Murakami, WB Saunders, Philadelphia, 1995, especially the 13rd page.

Term " prodrug " refers to that for the application the time to compare the cytotoxicity of cancerous cell less and can the enzymatic activation or change precursor and the derivative form of the active medicinal matter that has more active female medicine form into female medicine (parent drug).Referring to for example Wilman, " Prodrugs in Cancer Chemotherapy ", Biochemical Society Transactions, 14, pp.375-382,615th Meeting Belfast (1986) and Stella et al., " Prodrugs:A Chemical Approach to Targeted Drug Delivery ", Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985).Prodrug of the present invention includes but not limited to phosphate-containing/ester prodrug, containing sulfo-phosphate/ester prodrug, containing sulfate/ester prodrug, containing the peptide prodrug, the amino acid modified prodrug of D-, the glycosylation prodrug, containing the beta-lactam prodrug, containing the prodrug of optional substituted benzene oxygen yl acetamide or containing the prodrug of optional substituted benzene acetamide, can be converted into and have more activity and 5-flurocytosine and other 5-FUD prodrug of the medicine of no cytotoxicity.The example that can derive the cytotoxic drug of the prodrug form of using for the present invention includes but not limited to those chemotherapeutics described below.

The material that term " cytotoxic agent " refers to suppress or stop the function of cell and/or causes cytoclasis for this paper the time.This term intention comprises radiosiotope (At for example ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³²radiosiotope with Lu), the enzyme of chemotherapeutics and toxin such as micromolecule toxin or antibacterial, fungus, plant or the animal origin toxin of living, comprise its fragment and/or variant.

" chemotherapeutics " refers to can be used for the chemical compound for the treatment of as illness such as cancers.The example of chemotherapeutics comprises alkylating agent class (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and endoxan (cyclophosphamide) (CYTOXAN ^TM); Alkyl sulfonate esters class (alkyl sulfonates), such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan); Aziridines (aziridines), such as Benzodepa (benzodepa), card ripple quinone (carboquone), U.S. appropriate in sending (meturedepa) and uredepa (uredepa); Ethylenimine class (ethylenimines) and methylmelamine class (methylamelamines), comprise hemel (altretamine), triethylenemelamine (triethylenemelamine), APO (triethylenephosphoramide), TESPA (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine); Annonaceousacetogenicompounds (acetogenin) (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)); Camptothecine (camptothecin) (comprising synthetic analogues Hycamtin (topotecan)); Bryostatin (bryostatin); Callystatin; CC-1065 (comprising its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues); Hidden algae element class (cryptophycins) (particularly hidden algae element 1 and hidden algae element 8); Dolastatin (dolastatin); Duocarmycin (comprising synthetic analogues, KW-2189 and CB1-TM1); Eleutherobin (eleutherobin); Pancratistatin; Sarcodictyin; Sponge chalone (spongistatin); Nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlomaphazine), courage phosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine),Mustron (mechlorethamineoxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard), nitrosourea (nitrosoureas), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine), Ranimustine (ranimustine), antibiotics, for example, such as Enediyne Antibiotic (enediyne) (Calicheamicin (calicheamicin), especially Calicheamicin γ ₁ ^Iwith Calicheamicin ω ^I ₁(referring to for example Agnew, Chem.Intl.Ed.Engl.33:183-186 (1994)), dynemicin comprises dynemicin A, Ai Sibo mycin (esperamicin), and Neocarzinostatin (neocarzinostatin) chromophore and related colour albumen Enediyne Antibiotic chromophore), aclacinomycin (aclacinomycin), D actinomycin D (actinomycin), anthramycin (authramycin), azaserine (azaserine), bleomycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6-phenodiazine-5-oxygen-L-nor-leucine, Doxorubicin (doxorubicin) (comprises the morpholino Doxorubicin, cyano group morpholino Doxorubicin, 2-pyrroles is for Doxorubicin and deoxidation Doxorubicin), epirubicin (epirubicin), esorubicin (esombicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins), mycophenolic acid (mycophenolic acid),Nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), potfiromycin, puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin); The antimetabolite class, such as methopterin (methotrexate) and 5 FU 5 fluorouracil (5-FU); Folacin, such as denopterin (denopterin), methopterin (methotrexate), pteroyltriglutamic acid (pteropterin), Trimetrexate (trimetrexate); Purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine); Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, Carmofur (carmofur), cytarabine (cytarabine), two BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; Androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone); Anti-adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane); Folic acid supplement, such as folinic acid (folinic acid); Aceglatone (aceglatone); Aldophosphamide glucosides (aldophosphamide glycoside); Amino-laevulic acid (aminolevulinicacid); Amsacrine (amsacrine); Bestrabucil; Bisantrene (bisantrene); Edatrexate (edatraxate); Defosfamide (defosfamide); Demecolcine (demecolcine); Diaziquone (diaziquone); Elfomithine;Elliptinium Acetate (elliptinium acetate); Epothilones (epothilone); Ethoglucid (etoglucid); Gallium nitrate; Hydroxyl urea (hydroxyurea); Lentinan (lentinan); Lonidamine (lonidamine); Maytansinoid class (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin); Mitoguazone (mitoguazone); Mitoxantrone (mitoxantrone); Mopidamol (mopidamol); C-283 (nitracrine); Pentostatin (pentostatin); Phenamet (phenamet); THP (pirarubicin); Podophyllic acid (podophyllinic acid); 2-ethyl hydrazides (ethylhydrazide); Procarbazine (procarbazine); PSK

Razoxane (razoxane); Rhizomycin (rhizoxin); Sizofiran (sizofiran); Spirogermanium (spirogermanium); Tenuazonic acid (tenuazonic acid); Triethyleneiminobenzoquinone (triaziquone); 2,2 ', 2 " RA3s; Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and the rhzomorph (anguidin) that crawls); Urethane (urethan); Eldisine (vindesine); Dacarbazine (dacarbazine); Mannomustin (mannomustine); Dibromannitol (mitobronitol); Mitolactol (mitolactol); Pipobroman (pipobroman); Gacytosine; Cytarabine (arabinoside) (" Ara-C "); Endoxan (cyclophosphamide); Phosphinothioylidynetrisaziridine (thiotepa); Taxoid class (taxoids),Taxol (paclitaxel) (TAXOL for example

, Bristol-Myers Squibb Oncology, Princeton, NJ) and many Xi Taji (doxetaxel) (TAXOTERE , Rh

Ne-Poulenc Rorer, Antony, France); Chlorambucil (chlorambucil); Gemcitabine (gemcitabine); 6-thioguanine (thioguanine); Purinethol (mercaptopurine); Methopterin (methotrexate); Platinum analogs, such as cis-platinum (cisplatin) and carboplatin (carboplatin); Vincaleukoblastinum (vinblastine); Platinum; Etoposide (etoposide) (VP-16); Ifosfamide (ifosfamide); Mitomycin (mitomycin) C; Mitoxantrone (mitoxantrone); Vincristine (vincristine); Vinorelbine (vinorelbine); NVB (navelbine); NSC-279836 (novantrone); Teniposide (teniposide); Daunomycin (daunomycin); Aminopterin (aminopterin); Xeloda (xeloda); Ibandronate (ibandronate); CPT-11; Topoisomerase enzyme inhibitor RFS2000; DFMO (DMFO); Retinoic acid (retinoic acid); Capecitabine (capecitabine);And pharmaceutically acceptable salt, acid or the derivative of any above-mentioned substance.This definition also comprise act as regulate or inhibitory hormone to the antihormone agent of function of tumor, such as anti-estrogens, comprise that for example tamoxifen (tamoxifen), raloxifene (raloxifene), 4 (5)-imidazoles that suppress aromatase, 4-hydroxytamoxifen, trioxifene (trioxifene), that Lip river former times fragrant (keoxifene), LY117018, onapristone (onapristone) and toremifene (toremifene) are (Fareston); And anti-androgens, such as Drogenil (flutamide), nilutamide (nilutamide), than Ka meter Te (bicalutamide), leuprorelin (leuprolide) and goserelin (goserelin); And pharmaceutically acceptable salt, acid or the derivant of any above-mentioned substance.

Term " cytokine " " be to be discharged by a kind of cell mass, act on the common name of the protein of another cell as the iuntercellular medium.The example of this type cytokines has lymphokine, monokine and traditional polypeptide hormone.Cytokine comprises growth hormone, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; Insulin; Proinsulin; Relaxins; Relaxins is former; Glycoprotein hormone, such as follicle stimulating hormone (FSH), thyrotropin (TSH) and metakentrin (LH); Liver growth factor; Fibroblast growth factor; Prolactin antagonist; Human placental lactogen; Tumor necrosis factor-alpha and-β; Mu Leshi (Mullerian) inhibitory substance; Mice promoting sexual gland hormone related peptides; Inhibin; Activin; VEGF; Integrin; Thrombopoietin (TPO); Nerve growth factor, such as NGF-α; Platelet derived growth factor; Transforming growth factor (TGF), such as TGF-α and TGF-β; Insulin like growth factor-1 and-II; Erythropoietin (EPO); Bone-inducing factor (osteoinductive factor); Interferon, such as interferon-' alpha ' ,-β and-γ; Colony stimulating factor (CSF), such as macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF); Interleukin (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; Tumor necrosis factor, such as TNF-α or TNF-β; And other polypeptide factor, comprise LIF and kit part (KL).For this paper the time, the term cytokine comprises from natural origin or from the protein of recombinant cell culture thing and the biologic activity equivalent of native sequences cytokine.

Term " processing ", " treatment " and " therapy " refer to therapeutic treatment, preventative processing and precaution processing for this paper the time.

Term " treatment effective dose " refers to effectively treat disease or disorderly medication amount in mammal.In the situation of cancer, the treatment effective dose of medicine can reduce the number of cancerous cell; Dwindle the size of tumor; Suppress (slow down to a certain extent and preferably stop) cancer cell infiltration in peripheral organs; Suppress (slow down to a certain extent and preferably stop) neoplasm metastasis; Suppress to a certain extent tumor growth; And/or alleviate to a certain extent one or more symptoms relevant with disease.Can stop growth and/or kill on the degree of existing cancerous cell at medicine, it can be the inhibition cell and/or Cytotoxic.For cancer therapy, can and/or measure the speed of response (RR) and measure effect in body by for example assess tumor burden or volume, progression of disease time (TTP).

Term " mammal " aim for this paper the time enters mammiferous any mammal, comprises the people, High Primates, cattle, horse, dog and cat.In a preferred embodiment of the invention, mammal refers to the people.

Term " objective response " (objective response) is defined as the wholly or in part response of experimenter to treatment, use solid tumor response evaluation criteria (Response Evaluation Criteria in Solid Tumors, RECIST) (J.Nat.Cancer hist.92 (3): 205-216 (2000)).

Term " persistent period of response " (duration of response) was used in reference to from initial response or partial response in this article to disease progression or time when dead.

Term " progresson free survival " is used in reference in this article from treating the time of first day to disease progression or death (first sending out the survivor in both).

Term " (CR) fully disappears " is for showing the continuous gross tumor volume≤13.5mm measured for three times ³.

Term " part disappear (PR) " shows 50% and these one or many>=13.5mm in measuring of the continuous gross tumor volume of measuring for three times≤its first day volume ³.

II. the compositions and methods of the invention

a.DR5 antibody

In one embodiment of the invention, provide DR5 antibody.Exemplary antibody comprises polyclonal, monoclonal, humanized, bispecific and the antibody allos coupling.These antibody can be agonist, antagonist or blocking antibody.

1. polyclonal antibody

Antibody of the present invention can comprise polyclonal antibody.Method for the preparation of polyclonal antibody is well known by persons skilled in the art.Polyclonal antibody can generate in mammal, for example, by the agent of one or many injecting immune and adjuvant as required.Usually, immunizing agent and/or adjuvant will be expelled in mammal by subcutaneous or peritoneal injection repeatedly.Immunizing agent can comprise DR5 polypeptide (or DR5 ECD) or its fusion rotein.By immunizing agent and known immunogenic protein coupling to be arranged in immune mammal may be useful.The example of this type of immunogenic protein includes but not limited to keyhole

hemocyanin, serum albumin, bovine thyroglobulin and soybean trypsin inhibitor.The example of adoptable adjuvant comprises Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).Immunization protocol can be by those skilled in the art without too much testing and make a choice.Then can take a blood sample to mammal, and to determination of serum DR5 antibody titer.As required, can carry out booster immunization to mammal, until titre raises or reaches stable high concentration (plateau).

2. monoclonal antibody

Perhaps, antibody of the present invention can be monoclonal antibody.Monoclonal antibody can be used the hybridoma legal system standby, and such as Kohler and Milstein, Nature256:495 (1975) is described.In the hybridoma method, mice, hamster or other suitable host animal maybe can generate the lymphocyte of the antibody of specific binding immunizing agent to cause to generate with the immunizing agent immunity usually.Perhaps, immunological lymphocyte in vitro.

Immunizing agent will generally include DR5 polypeptide (or DR5 ECD) or its fusion rotein, such as the DR5ECD-IgG fusion rotein.Perhaps, immunizing agent can comprise fragment or the part of DR5, wherein has one or more aminoacid to participate in the combination of Apo-2L and DR5.In a preferred embodiment, immunizing agent comprises the DR5 ectodomain sequence merged with the IgG sequence.

Usually, if wish, for the cell of people's origin, use peripheral blood lymphocyte (" PBL "),, or, splenocyte or lymph-node cell used if wish for the cell in non-human mammal source.Then, use suitable fusion agent, such as Polyethylene Glycol, lymphocyte and immortalized cell line are merged to form hybridoma (Goding, Monoclonal Antibodies:Principles and Practice, AcademicPress (1986), pp.59-103).The myeloma cell of the mammalian cell that immortalized cell line normally transforms, particularly Rodents, cattle and people's origin.Usually, adopt rat or mouse myeloma cell line.Hybridoma can be cultivated in suitable culture medium, and described medium optimization contains immortalized cells growth that inhibition do not merge or one or more materials of survival.For example, if parental cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), culture medium for hybridoma will contain hypoxanthine, aminopterin-induced syndrome and thymidine (" HAT culture medium ") usually so, and these materials stop the growth of HGPRT deficient cells.

Preferred immortalized cell line is that those efficiently merge, support selected antibody-producting cell stably to express antibody and to the cell line such as culture medium sensitivities such as HAT culture medium high-levelly.Preferred immortalized cell line is Mus source myeloma system, can be from for example Sol gram institute cell distributing center (SalkInstitute Cell Distribution Center, San Diege, California, USA) and American type culture collection (American Type Culture Collection, Manassas, Virginia, USA) obtain.An example of this type of rat bone marrow tumour cell system is P3X63Ag8U.1 (ATCC CRL1580).Also have description (Kozbor, J.Immunol.133:3001 (1984) for human myeloma and the mice-people's allos myeloma cell line that generates human monoclonal antibodies; Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, Marcel Dekker, Inc., New York (1987) pp.51-63).

Then the culture medium that can cultivate therein hybridoma is measured the existence for the monoclonal antibody of DR5.Preferably, by immunoprecipitation or by external binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), measure the binding specificity of the monoclonal antibody generated by hybridoma.This type of technology and algoscopy are known in the art.The binding affinity of monoclonal antibody can be by for example Munson and Pollard, and the Scatchard of Anal.Biochem.107:220 (1980) analyzes to measure.

Obtain the hybridoma of expectation in evaluation after, this clone can carry out sub-clone by the limiting dilution flow process, and is cultivated (Goding sees above) by standard method.The culture medium that is suitable for this purpose comprises EagleShi culture medium or the RPMI-1640 culture medium of for example DulbeccoShi improvement.Perhaps, hybridoma can carry out culturing in vivo as ascites in mammal.

Can pass through routine immunization globulin purification flow process, such as for example protein A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatograph, the monoclonal antibody of sub-clone secretion be separated or purification with culture medium or ascites.

Monoclonal antibody also can generate by recombinant DNA technology, such as United States Patent (USP) 4,816, described in 567.The DNA of coding monoclonal antibody is easy to use old process to separate and order-checking (for example using can specific binding coding monoclonal antibody heavy chain and the oligonucleotide probe of the gene of light chain).Hybridoma is the preferred source of this type of DNA.Once separate, DNA can be placed in to expression vector, then this expression vector is transfected in the host cell that does not produce in addition immunoglobulin protein, such as Bacillus coli cells, ape and monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthetic of monoclonal antibody in recombinant host cell.All right modifying DNA, for example, by substituting, the coded sequence that is employment heavy chain and constant region of light chain replaces homology Mus source sequence (Morrison et al., or engage the coded sequence all or in part of immunoglobulin coding sequence and NIg polypeptide by covalency Proc.Nat.Acad.Sci.81:6851 (1984)).Can prepare and there is " chimeric " or " heterozygosis " antibody of the binding specificity of anti-DR5 monoclonal antibody herein in this mode.

Usually substitute the constant region of antibody of the present invention with this type of NIg polypeptide, the variable region of an antigen binding site that perhaps with their, substitutes antibody of the present invention to be to produce chimeric bivalent antibody, and it comprises to DR5 is had a specific antigen binding site and synantigen is not had to specific another antigen binding site.

Also can prepare with the known method of synthetic protein chemistry by chimeric or hybrid antibody, comprise that those relate to the method for cross-linking agent in vitro.For example, can build immunotoxin with the disulfide exchange reaction or by forming thioether bond.The example that is suitable for the reagent of this purpose comprises imino group mercaptan ester/salt (iminothiolate) and 4-sulfydryl butyryl imidic acid methyl ester (methyl-4-mercaptobutyrimidate).

Also can generate Single-Chain Fv Fragment of Murine, such as Iliades et al., FEBS Letters, described in 409:437-441 (1997).This type of single-chain fragment is used the coupling of various terminal to be recorded in Kortt et al., ProteinEngineering, 10:423-433 (1997).The multiple technologies for recombinant production and operation antibody are known in this area.Hereinafter more detailed description the il-lustrative example of normally used these type of technology of those of skill in the art.

(i) humanized antibody

Usually, one or more amino acid residues from inhuman source have been introduced in humanized antibody.These inhuman amino acid residues are often referred to as " input " residue, and they take from " input " variable region usually.Humanization can basically be followed Winter and colleague's thereof method and carry out (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), use the corresponding sequence of Rodents CDR or CDR sequence replacing people antibody.

Therefore, this type of " humanization " antibody is chimeric antibody, wherein basically is less than whole people variable region and uses the corresponding sequence from inhuman species to substitute.In practice, normally some of them CDR residue and possible some FR residues people antibody alternative from the residue in similar site in Rodents antibody of humanized antibody.

Importantly, antibody keeps high-affinity and other the favourable biological characteristics to antigen after humanization.In order to realize this goal, according to a kind of preferred method, the method for analyzing parental array and each ways makes conceptual researches humanization product by the threedimensional model by parent and humanization sequence prepares humanized antibody.Three-dimensional immunoglobulin model is normally obtainable, and is familiar with by those skilled in the art.Can obtain the computer program of the possible three-dimensional conformation structure of diagram and the selected candidate's immunoglobulin sequences of demonstration.Check that these displayed map look like to allow analysis residue may act in candidate's immunoglobulin sequences functionating, analyzing influence candidate immunoglobulin is in conjunction with the residue of the ability of its antigen.Like this, can from total and list entries, select the FR residue and be combined, thereby obtain expectation antibody feature, improve such as the affinity to target antigen.Generally speaking, the CDR residue directly and essence relate to the impact on the antigen combination.

(ii) people's antibody

Human monoclonal antibodies can generate by hybridoma method.For generating, the human myeloma of human monoclonal antibodies and mice-people's allos myeloma cell line are existing to be described, Kozbor for example, J.Immunol.133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63, Marcel Dekker, Inc., New York (1987).

Now likely be created on the transgenic animal (for example mice) that can when immunity, generate the complete complete or collected works of people's antibody in the situation that lacks endogenous immunoglobulin generation.For example, put down in writing heavy chain of antibody bonding pad (J in chimeric and germ line mutation mice _h) isozygotying of gene delete the inhibition fully cause endogenous antibody to generate.Shift a large amount of people's germline immunoglobulin genes and will cause generating people's antibody in this type of germ line mutation mice when antigen is attacked.Referring to for example Jakobovits et al., Proc.Natl.Acad.Sci.USA90:2551-255 (1993); Jakobovits et al., Nature362:255-258 (1993).

The people such as Mendez (Nature Genetics15:146-156 (1997)) have further improved technology, generated the transgenic mice strain that is called " Xenomouse II " (xenograft mice), it is at the antibody that is subject to when antigen is attacked generating the complete people of high-affinity.This is to be incorporated into endogenous J by the people's heavy chain by millions of bases and light chain gene seat germline as mentioned above _hsection has in the mice of deletion and realizes.Xenomouse II carries people's heavy chain gene seat of 1,020kb, comprises about 66 kinds of V _hgene, complete D _hand J _hdistrict, and three kinds of different constant regions (μ, δ and χ), also carry 800kb people's kappa gene seat, comprises 32 kinds of V kappa genes, J κ section and C kappa gene.The antibody generated in these mices in all respects in human body, see extremely similar, comprise gene rearrangement, assembling and complete or collected works.Due to endogenous J _hthe deletion of section has prevented the gene rearrangement of musculus cdna seat, so people's antibody is preferentially expressed than endogenous antibody.

Perhaps, display technique of bacteriophage (McCafferty et al., Nature348:552-553 (1990)) is used in external from from the immune globulin variable region of epidemic disease donor (V) gene complete or collected works rather, generating people's antibody and antibody fragment.According to this technology, antibody V domain gene is cloned in the main or less important coat protein gene of filobactivirus such as M13 or fd in the mode that meets reading frame, and is shown as the functional antibodies fragment on the phage particle surface.Because the single stranded DNA that the filobactivirus granule comprises phage genome copy, the selection that the functional characteristic of antibody of take carries out as basis also causes coding to show the selection of gene of the antibody of those characteristics.So, some characteristics of phage simulation B cell.Phage display can carry out in a variety of forms; Relevant summary is referring to for example Johnson, Kevin S.and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V constant gene segment C can be used for phage display.Clackson et al., Nature352:624-628 (1991) separates and obtains a large amount of different anti-oxazolone antibody from the small-sized V gene random combine library derived from through the immune mouse spleen.Can follow in essence Marks et al., J.Mol.Biol.222:581-597 (1991) or Griffith et al., EMBO is (1993) described technology J.12:725-734, by not immune people's donor, builds V gene complete or collected works and Separated pin to the antibody of synantigen (comprising autoantigen) not in a large number.In innate immunity, antibody gene is with height ratio accumulation sudden change (somatic hypermutation).Some variation imported will be given more high-affinity, show that the B cell of high-affinity surface immunoglobulin preferentially copies and breaks up in antigen attack process subsequently.This natural process can be called by employing the technology of " chain reorganization " and simulate (Marks et al., Bio/Technol.10:779-783 (1992)).In this method, the affinity of " initially " the people's antibody obtained by phage display can be replaced heavy chain and light chain V district gene improves in succession by the naturally occurring variant of V domain gene (complete or collected works) with never immune donor obtains.This technology is generated antibody and the antibody fragment of affinity in the nM scope.Waterhouse et al., Nucl.Acids Res.21:2265-2266 (1993) has put down in writing for building the strategy of very large phage complete or collected works (also referred to as " Mu storehouse, all libraries " (the mother-of-all libraries)).Gene shuffling also can be used for from the derivative people's antibody of Rodents antibody, and wherein people's antibody has affinity and the specificity similar to initial Rodents antibody.According to the method, it is also referred to as " the epi-position marking " (epitope imprinting), and the heavy chain of the Rodents antibody obtained by display technique of bacteriophage or light chain V domain gene employment V domain gene complete or collected works replace, and produce Rodents-people's block polymer.The selection that antigen is carried out causes the separation of people variable region that can the restore functionality antigen binding site, and epi-position determines (marking, imprint) selection of gametophyte.When repeating this process with replacement residue Rodents V domain, obtain people's antibody (referring to PCT patent application WO93/06213, being disclosed on April 1st, 1993).From traditional to transplant the humanization of the Rodents antibody carried out by CDR different, this technology provides complete people's antibody, and they are containing framework or the CDR residue of Rodents origin.

As what hereinafter discuss in detail, antibody of the present invention can optionally comprise the multivalence form of antibody monomer, antibody dimer and antibody.Those skilled in the art can build this type of dimer or multivalence form by technology known in the art and with DR5 antibody herein.Method for the preparation of univalent antibody is also well-known in the art.For example, a kind of method relates to the recombinant expressed of light chain immunoglobulin and modified heavy chain.Heavy chain is usually crosslinked to prevent heavy chain in the Zhong arbitrfary point truncate of Fc district.Perhaps, relevant cysteine residues substitutes with another kind of amino acid residue or deletes crosslinked to prevent.

(iii) bi-specific antibody

Bi-specific antibody refer to at least two kinds not synantigen there is the monoclonal of binding specificity, preferably people or humanized antibody.In this case, a kind of binding specificity is to the DR5 receptor, and another kind is to any other antigen, preferably to another kind of receptor or receptor subunit.For example, the bi-specific antibody of specific binding DR5 receptor and another kind of apoptosis/frizzled receptor within the scope of the invention.

For the method that generates bi-specific antibody, be known in the art.Traditionally, the restructuring of bi-specific antibody generates the coexpression based on two pairs of heavy chain immunoglobulin-light chains, and wherein two kinds of heavy chains have different specificity (Millstein and Cuello, Nature305:537-539 (1983)).Due to the random assortment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of ten kinds of different antibodies molecules, wherein only have a kind of correct bispecific structure that has.The suitable trouble of the purification of the correct molecule usually undertaken by the affinity chromatograph step and products collection efficiency are low.On May 13rd, 1993 disclosed WO93/08829 and Traunecker et al., EMBO 10:3655-3659 has disclosed similar flow process in (1991).

According to a kind of different and preferred method, antibody variable region and the constant region for immunoglobulin sequence that will have expectation binding specificity (antibody-antigen binding site) merge.Merge and preferably use the immunoglobulin heavy chain constant region that comprises at least part of hinge, CH2He CH3 district.Preferably at least one fusions, exist and comprise first CH (CH1) of light chain in conjunction with necessary site.The heavy chain immunoglobulin fusions of encoding reaches, and if necessary, the DNA of light chain immunoglobulin inserts expression vector separately, and cotransfection is in suitable host organisms.The embodiment of optimum point of production is provided when the three peptide species chain ratios for building do not wait, and this provides great motility for the mutual ratio of adjusting three peptide species fragments.Yet, at least two peptide species chains, with same ratio, express while causing high yield or when this ratio does not have special meaning, likely the coded sequence of two kinds or all three peptide species chains is inserted to same expression vector.In a preferred embodiment of the method, bi-specific antibody is by having the heterozygosis heavy chain immunoglobulin of the first binding specificity on an arm, and the heterozygosis heavy chain immunoglobulin-light chain on another arm forms (the second binding specificity is provided).Due to light chain immunoglobulin only the existence in half bispecific molecule the convenient approach separated is provided, therefore find this dissymmetrical structure be convenient to will expectation the bispecific complex combine and separate with undesired immunoglobulin chain.The method is disclosed in disclosed PCR application on March 3rd, 1994 WO94/04690.

About the further details that generate bi-specific antibody referring to for example Suresh et al., Methods inEnzymology121:210 (1986).

(iv) allos coupling antibody

Allos coupling antibody also within the scope of the invention.Allos coupling antibody consists of two kinds of covalently bound antibody.The suggestion of this antibody-like is for example for by the undesired cell of immune system cell targeting (United States Patent (USP) 4,676,980) and be used for the treatment of HIV and infect (PCT application WO91/00360 and WO92/200373; EP03089).Allos coupling antibody can generate with any cross-linking method easily.Suitable cross-linking agent is well-known in the art, together with many crosslinking technologicals, is disclosed in United States Patent (USP) 4,676,980.

(v) antibody fragment

In certain embodiments, Anti-DR5 antibody (comprise Mus, the people and humanized antibody, and antibody variants) be antibody fragment.Developed for generating the multiple technologies of antibody fragment.Traditionally, by the proteolytic digestion complete antibody derive these fragments (referring to for example Morimoto et al., J.Biochem.Biophys.Methods24:107-117 (1992); Brennan et al., Science229:81 (1985)).Yet, can directly by recombinant host cell, generate these fragments now.For example, can be directly from escherichia coli, reclaim Fab '-SH fragment chemical coupling to form F (ab ') ₂fragment (Carter et al., Bio/Technology10:163-167 (1992)).In another embodiment, F (ab ') ₂fragment is to use leucine zipper GCN4 to form, to promote F (ab ') ₂the assembling of molecule.According to another kind of method, can directly from the recombinant host cell culture, separate Fv, Fab or F (ab ') ₂fragment.For the multiple technologies that generate antibody fragment, to skilled practitioner, will be apparent.For example, can use papain to be digested.The example of papain digestion is recorded in 22, disclosed WO94/29348 of 94 on Decembers and United States Patent (USP) 4,342,566.Produce two identical Fabs with papain digestion antibody, be called the Fab fragment, there is separately an antigen binding site, and a remaining Fc fragment.Pepsin produces a F (ab ') ₂fragment, it has two antigen binding sites and still can crosslinked antigen.

The Fab fragment produced in antibody digestion also comprises the constant region of light chain and the first constant region (CH of heavy chain ₁).The difference of Fab ' fragment and Fab fragment is heavy chain CH ₁the carboxyl terminal of domain has increased the minority residue, comprises the one or more cysteine from antibody hinge region.Fab '-SH is herein to appellation that wherein the constant region cysteine residues carries the Fab ' of free sulphur alcohol radical.F (ab ') ₂antibody fragment is to generate as the paired Fab ' fragment that hinge cysteine is arranged between Fab ' fragment at first.Also know other chemical coupling of antibody fragment.

(vi) the aminoacid sequence variant of antibody

The aminoacid sequence variant of Anti-DR5 antibody changes and introduces Anti-DR5 antibody DNA or synthesize to prepare by peptide by the nucleotide by suitable.This type of variant comprises that in this paper embodiment for example, the residue in the Anti-DR5 antibody aminoacid sequence is deleted and/or inserts and/or substitutes.As long as final construction has desired characteristic, can be deleted, be inserted and alternative combination in any to obtain final construction.Aminoacid changes the translation post-treatment that also can change humanization or variation Anti-DR5 antibody, such as the number or the position that change glycosylation site.

Can be used for identifying in Anti-DR5 antibody being called " alanine scanning mutagenesis " as some residue of preferred mutation position or a kind of method in zone, as Cunningham and Wells, Science244:1081-1085 (1989) is described.Here, identified a residue or one group of target residue (charged residue for example, such as arginine, aspartic acid, histidine, lysine and glutamic acid), and substitute with neutral or electronegative aminoacid (most preferably alanine or polyalanine), to affect the interaction of aminoacid and DR5 antigen.Then pass through or alternate site is introduced to more or other variant, hammering out those to substituting the amino acid position that shows function sensitive.So, although introduce the site of variant amino acid sequence, predetermine, the character of sudden change itself does not need to predetermine.For example, in order to analyze the consequence in the sudden change of given site, at target codon or zone, carry out Alanine-scanning or random mutagenesis, and active to expressed Anti-DR5 antibody variant screening expectation.

Aminoacid sequence inserts the fusion comprise amino and/or carboxyl terminal, and length range is from a residue to the polypeptide that comprises up to a hundred or more residues, and inserts in the sequence of single or multiple amino acid residues.The example that end inserts comprises the Anti-DR5 antibody with N-end methionyl residue or the antibody merged with epitope tag.Other of Anti-DR5 antibody molecule inserts variant and is included in the N-of Anti-DR5 antibody or the enzyme that the C-end merges or polypeptide or the polyhydric alcohol that improves the antibody serum half-life.

Another kind of variant is the amino acid replacement variant.These variants have at least one amino acid residue remove and insert different residues in its position in the Anti-DR5 antibody molecule.The most interested site that substitutes mutation comprises hypervariable region, but has also imagined the FR change.Conservative substituting is displayed in Table 1 under title " preferably substitutes ".If this type of substitutes the change cause biologic activity, can in introducing table 1, be called so the more material alterations of " illustration substitutes ", or as hereinafter about the further describing of aminoacid kind, and screening product.

Table 1

Original residue	Illustration substitutes	Preferably substitute
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; Val; Met; Ala; Phe; Nor-leucine	Leu
			Leu(L)	Nor-leucine; Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala; Nor-leucine	Leu

The substance of antagonist biological characteristics is modified by being chosen in remarkable different alternative completing on the effect that keeps following aspect: (a) structure of the polypeptide main chain of replacement area, for example, as pleated sheet or helical conformation, (b) target site is punished sub electric charge or hydrophobicity, or (c) volume of side chain.Side chain characteristic based on common, the natural residue that exists can be divided into groups as follows:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: Cys, Ser, Thr;

(3) acid: Asp, Glu;

(4) alkalescence: Asn, Gln, His, Lys, Arg;

(5) affect the residue of chain orientation: Gly, Pro; And

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitute will need to replace another classification with a member in one of these classifications.

Any not relating to, keep the cysteine residues of humanization or the correct conformation of variation Anti-DR5 antibody also alternative, usually uses serine, with the oxidation stability of improving molecule with prevent extremely crosslinked.On the contrary, can in antibody, add the cysteine key to improve its stability (particularly when antibody is antibody fragment such as Fv fragment).

A particularly preferred class alternative variations relates to one or more hypervariable regions residue of alternative parental antibody (for example humanization or people's antibody).Usually, select will there is improved biological characteristics for the gained variant of further exploitation with respect to the parental antibody that produces them.A kind of facilitated method that produces this type of alternative variations is to use the affinity maturation of phage display.Briefly, for example, by site, several hypervariable region (6-7 site) sudden change, in each site, produce all possible amino acid replacement.The antibody variants so produced is illustrated on the filobactivirus granule with the unit price form, as the fusions of the M13 gene III product with each granule inner packing.Then for example, according to its biologic activity (binding affinity), the variant to phage display is screened as disclosed herein.In order to identify the site, candidate hypervariable region for modifying, can carry out alanine scanning mutagenesis to identify antigen in conjunction with the hypervariable region residue with significant contribution.Perhaps/in addition, analyze the crystal structure of antigen-antibody complex, to identify that the contact point between antibody and people DR5 may be useful.This type of contact residues and contiguous residue are to carry out alternative candidate locus according to technology detailed in this article.Once produce this type of variant, as described herein this group variant is screened, can be chosen in there is good characteristic in one or more related assays methods antibody for further exploitation.

(vii) glycosylation variants of antibody

Glycosylation (Jefferis and Lund, Chem.Immunol.65:111-128 (1997) occur in the conservative position of antibody in its constant region; Wright and Morrison, TibTECH15:26-32 (1997)).The oligosaccharide side chain of immunoglobulin affects function (Boyd et al., the Mol.Immunol.32:1311-1318 (1996) of protein; Wittwe and Howard, Biochem.29:4175-4180 (1990)), and the intramolecular interaction between the glycoprotein each several part, it can affect the conformation of glycoprotein and the three-dimensional surface presented (Hefferis and Lund, supra; Wyss and Wagner, Current Opin.Biotech.7:409-416 (1996)).Oligosaccharide also can be used to make given glycoprotein targeting some molecule based on the specific identification structure.For example, have been reported, in without galactosylation IgG, the oligosaccharide module is stretched out from the space between CH2, end N-acetyl-glucosamine residue is able in conjunction with mannose-binding protein (Malhotra et al., Nature Med.1:237-243 (1995)).The oligosaccharide of removing on the CAMPATH-1H (a kind of recombinant humanized Mus monoclonal IgG1 antibody of identifying human lymphocyte CDw52 antigen) generated in Chinese hamster ovary (CHO) cell with glycopeptidase causes the cytolysis (CMCL) of complement-mediated to reduce (Boyd et al. fully, Mol.Immunol.32:1311-1318 (1996)), eliminate with the neuraminic acid enzyme selectivity forfeiture that sialic acid residues does not cause DMCL.Also have report, the glycosylation of antibody affects the cytotoxicity of antibody dependent cellular (ADCC).Particularly, report, wherein β (1,4) expression of-N-acetyl-glucosamine transferase I II (GnTIII) (glycosyl transferase that minute GlcNAc such as catalysis forms) is subject to the Chinese hamster ovary celI of tetracycline regulation and control to have the ADCC active (Umana et al., Mature Biotech.17:176-180 (1999)) of improvement.

The glycosylation variants of antibody refers to the variant that wherein the glycosylation pattern of antibody changes.Change and to mean and delete one or more carbohydrate modules of finding in antibody, in antibody, add one or more carbohydrate modules, change the composition of glycosylation (glycosylation pattern), glycosylated degree, etc.Glycosylation variants can be by for example eliminating in the nucleotide sequence of encoding antibody, change and/or add one or more glycosylation sites prepares.

That the glycosylation of antibody is typical or N-connects or the O-connection.N-connects and refers to that the carbohydrate module is attached to the side chain of asparagine residue.Tripeptide sequence agedoite-X-serine and agedoite-X-threonine (wherein X is any aminoacid except proline) is carbohydrate module enzymatic to be attached to the recognition sequence of agedoite side chain.So, in polypeptide, these two kinds of arbitrary existence of tripeptide sequence have produced potential glycosylation site.The glycosylation that O-connects refers to one of saccharide N-acetylgalactosamine, galactose or xylose are attached to hydroxy-amino-acid, and modal is serine or threonine, but also can use 5-OxoPro or 5-hydroxylysine.

Can make it comprise one or more above-mentioned tripeptide sequences and complete easily (glycosylation site connected for N-) by changing aminoacid sequence to adding glycosylation site in antibody.Described change also can be by adding in the sequence to initial antibodies or substituting one or more serines or threonine residues is carried out (glycosylation site connected for O-).

The several different methods preparation that the nucleic acid molecules of coding Anti-DR5 antibody aminoacid sequence variant can be known by this area.These methods include but not limited to separate (the natural situation that has an aminoacid sequence variant) from natural origin, or the Anti-DR5 antibody by the variant to early stage preparation or non-variant pattern carries out oligonucleotide mediated (or fixed point) mutation, PCR mutation and cassette mutagenesis and prepares.

Can also under the prerequisite that does not change potential nucleotide sequence, change the glycosylation (comprising the glycosylation pattern) of antibody.Glycosylation depends on to a great extent for expressing the host cell of antibody.Due to for express as the recombinant glycoprotein of potential therapeutic agent for example the cell type of antibody be seldom n cell, therefore can expect that the glycosylation pattern of antibody will have significant variation (referring to for example Hse et al., J.Biol.Chem.272:9062-9070 (1997)).Outside the selection of host cell, in the restructuring generative process of antibody, the glycosylated factor of impact comprises growth pattern, culture medium prescription, culture density, Oxygenation (oxygenation), pH, purification schemes, like that.Proposed several different methods for changing the glycosylation pattern of realizing at the specific host organism, comprised importing or cross to express relating to glycogenetic some enzyme of widow (United States Patent (USP) 5,047,335; 5,510,261 and 5.278,299).The glycosylation of glycosylation or some type can be removed by enzymatic from glycoprotein, for example uses endoglycosidase H (Endo H).In addition, can carry out genetic engineering reorganization to recombinant host cell, for example make its defect aspect the polysaccharide of some type of processing.These and similar technology are well-known in the art.

The glycosylation structure of antibody can be easy to by the routine techniques of carbohydrate analysis analyze, comprise agglutinin chromatography, NMR, mass spectrum, HPLC, GPC, monosaccharide component analysis, sequential enzymatic digestion and HPAEC-PAD, it utilizes high pH anion-exchange chromatography to separate oligosaccharide according to electric charge.The method that discharges oligosaccharide for analysis purpose also knows, include but not limited to enzymatic treatment (usually using peptide-N-glycosidase F/ inscribe-beta galactosidase to carry out), use the elimination of strong alkali environment to be mainly the O-syndeton to discharge, and the chemical method of using anhydrous hydrazine with discharge N-with O-, be connected oligosaccharide the two.

(viii) exemplary antibody

Invention disclosed herein has many exemplary embodiment.A plurality of typical embodiments of the present invention has hereinafter been described.It is for the illustration purpose that following embodiment is provided, and is not intended to limit the scope of the invention by any way.

Described in hereinafter embodiment, many Anti-DR5 antibodies have been identified.In one embodiment, the biological property that DR5 antibody of the present invention will have with clearly disclosed any Anti-DR5 antibody is identical herein.

Term " biological property " is used in reference to external and/or activity in vivo or the characteristic of monoclonal antibody, such as specific binding DR5 or blocking-up, induce or strengthen the ability of DR5 activation (or DR5 related activity).The characteristic of DR5 antibody and activity further describe in embodiment hereinafter.

Optional is, monoclonal antibody of the present invention will have the biological property identical with listed any antibody in antibody 16E2 or table 11-13, and/or with antibody 16E2 or table 11-13 in listed any antibody, particularly Apomab 7.3 or Apomab 8.3 in conjunction with identical epi-position.This can measure by carrying out the many measure method, described in this paper and embodiment.For example, in order to measure certain monoclonal antibody, whether there is the specificity identical with the DR5 antibody of clearly mentioning herein, can in competitive binding assay method or apoptosis induction algoscopy, compare its activity, such as the algoscopy described in embodiment hereinafter.In addition, the epi-position of specific Anti-DR5 antibody institute combination can be measured by DR5 and the crystallography research of complex between antibody of discussing.

So, between the ectodomain of the Fab fragment of Apomab 7.3 and DR5, the X-ray crystallography of complex studies show that out that Apomab 7.3 is in conjunction with the DR5 epi-position overlapping but different from the binding site of Apo2L/TRAIL on the DR5 receptor.

Anti-DR5 antibody (and for example double antibody described herein or the three antibody) people, chimeric, heterozygosis or restructuring can comprise the antibody with total length heavy chain and light chain, or its fragment, such as Fab, Fab ', F (ab ') ₂or the variable region (or hypervariable region) of the monomer of Fv fragment, described light chain or heavy chain or dimer, wherein said heavy chain or the light chain scFv connected by linkers or described light chain or the heavy chain scFv of combining with the antibody structure territory of other type.

As described herein, DR5 antibody will optionally have biologic activity or the characteristic of one or more expectation.This type of DR5 antibody can include but not limited to chimeric, humanized, the people and antibody affinity maturation.As mentioned above, DR5 antibody can be used multiple technologies to build or reorganize to realize these expectation activity or characteristics.In one embodiment, DR5 antibody will have at least 10 ⁵m ^-1dR5 receptors bind affinity, preferably at least 10 ⁶m ^-1to 10 ⁷m ^-1scope in, more preferably at least 10 ⁸m ^-1to 10 ¹²m ^-1scope in, even more preferably at least 10 ⁹m ^-1to 10 ¹²m ^-1scope in.Without too much experiment, the binding affinity of DR5 antibody can be measured by test DR5 antibody according to technology known in the art, comprises that Scatchard analyzes (referring to Munson et al., supra).

In another embodiment, DR5 of the present invention can with epi-position Apo-2L institute combination identical epi-position upper in conjunction with DR5, or in conjunction with upper epi-position coincidence or the overlapping epi-position of being combined with Apo-2L of DR5, as the antibody that for example is called Apomab 7.3.DR5 antibody can also a kind of like this mode interact, so that produce, prevents the space conformation of Apo2L/TRAIL in conjunction with DR5.As mentioned above, the epi-position binding characteristic of DR5 antibody of the present invention can be measured by technology known in the art.For example, test in vitro DR5 antibody in algoscopy, such as the competitive inhibition algoscopy, to measure the DR5 antibody blocking or to suppress the ability of Apo-2L in conjunction with DR5.Optional, can in the competitive inhibition algoscopy, test DR5 antibody to measure DR5 antibody suppression Apo-2L polypeptide in conjunction with the DR5-IgG construction or to express the ability of the cell of DR5.Optional is, DR5 antibody can or suppress at least 50% by the combination blocking-up of Apo-2L and DR5, preferably at least 75%, even more preferably at least 90%, this can for example use soluble form Apo2L/TRAIL (TRAIL) (such as Pitti et al., J.Biol.Chem., the ectodomain sequence 114-281 position of putting down in writing in supra, also referred to as Apo2L.0) and the external competitive inhibition algoscopy of DR5 ECD-IgG in measure.The external test method of the ability of the epi-position binding characteristic of the DR5 antibody apoptosis that also but use test DR5 antibody blocking Apo-2L induces or measure by crystallography research.

In another embodiment, DR5 antibody will comprise the agonistic antibody with activity suitable with Apo2L/TRAIL (TRAIL).Preferably, this type of excitability DR5 antibody will be apoptosis-induced in the cancer of at least one type or tumor cell line or primary tumor.The apoptosis activity of excitability DR5 antibody can be measured by known external or in vivoassay method.The example of multiple this type of in vitro and in vivo algoscopy is well-known in the art.In vitro, apoptosis activity can be measured with known technology, such as the annexin V combination.In vivo, apoptosis activity can by for example measure tumor load or volume dwindle measure.

As mentioned above, antibody disclosed herein has numerous characteristics, comprises that some physiology of regulation and control interacts and/or the ability of process.As shown in hereinafter embodiment, antibody disclosed herein can be induced the apoptosis of DR5 mediation, and demonstrates strong antitumor properties in the multiple Mus xenograft models of cancer.In a specific embodiments of the present invention, the excitability activity of antibody is by by antibody and crosslinked being enhanced of anti-human IgG Fc.In a preferred embodiment of the invention, the apoptosis activity of the apoptosis of this enhancing and Apo-2L is suitable.

Other embodiment of the present invention comprises so anti-DR5 receptor antibody disclosed herein, and it is connected with the polymer that is selected from one or more nonprotein character of lower group: Polyethylene Glycol, polypropylene glycol and polyoxyalkylene (polyoxyalkylene).In an alternate embodiment, anti-DR5 receptor antibody disclosed herein is connected with cytotoxic agent or enzyme.In also having an embodiment, anti-DR5 receptor antibody disclosed herein is connected with radiosiotope, fluorescent chemicals or chemiluminescence compound.Optional, anti-DR5 receptor antibody disclosed herein is glycosylated or not glycosylated.

As hereinafter discussed in detail, antibody of the present invention can be used for regulating and controlling the several different methods of physiological processes.Such embodiment of the present invention is included in method apoptosis-induced in mammalian cell, comprise that the mammalian cell that makes to express the DR5 receptor is exposed to the anti-DR5 acceptor monoclonal antibody of the separation for the treatment of effective dose, comprises in conjunction with DR5 receptor shown in Fig. 3 A-3C (411 aminoacid) or Fig. 4 A-4C (440 aminoacid) the especially antibody of its ectodomain.In these class methods, described mammalian cell is cancerous cell normally.In preferred embodiments, the anti-DR5 receptor antibody used in these methods is the Apomab antibody described in embodiment hereinafter, such as Apomab 7.3 or Apomab8.3 antibody.

An embodiment in addition of the present invention is method apoptosis-induced in mammalian cell, comprise that the mammalian cell that makes to express the DR5 receptor is exposed to the anti-DR5 acceptor monoclonal antibody of the separation for the treatment of effective dose, comprise in conjunction with the antibody of DR5 receptor or its ectodomain as defined above.

3 three chain antibodies

Three chain antibodies (triabody) also within the scope of the invention.This antibody-like is recorded in for example Iliades etal., supra and Kortt et al., supra.

4. other modification

Imagined other modification of DR5 antibody herein.Antibody of the present invention can be by modifying antibody and cytotoxic agent (as lps molecule) or the coupling of prodrug activation enzyme, described prodrug activation enzyme changes prodrug (for example peptidyl chemotherapeutics, referring to WO81/01145) into the active anticancer medicine.Referring to for example WO88/07378 and United States Patent (USP) the 4th, 975, No. 278.This technology also referred to as " relying on the prodrug therapy of the enzyme mediation of antibody " (ADEPT).

Thereby the enzyme component that can be used for the immune conjugate of ADEPT comprises that can act in such a way prodrug changes it any enzyme of more activated cytotoxicity form into.The enzyme that can be used for the inventive method includes but not limited to the prodrug of phosphate-containing/ester to be changed into to the alkali phosphatase of free drug; The prodrug of containing sulfate/ester can be changed into to the aryl sulfatase of free drug; Nontoxic 5-flurocytosine can be changed into to the cytosine deaminase of anticarcinogen 5-fluorouracil; Can be by change the protease of free drug into containing the propeptide medicine, such as Serratieae protease, thermolysin, subtilisin, carboxypeptidase and cathepsin (such as cathepsin B and L); Caspase, such as Caspase-3; Can transform the D-alanyl carboxypeptidase containing the prodrug of D-amino acid replacement; The glycosylation prodrug can be changed into to the carbohydrate nickase of free drug, such as beta galactosidase and neuraminidase; The medicine derivative with beta-lactam can be changed into to the beta-lactamase of free drug; And can be by be transformed into respectively the penicillin amidase of free drug with benzene oxygen acetyl group or the derivative medicine of phenylacetyl group at its amino nitrogen place, such as penicillin V amidase or Penicillin-G-amidases.Perhaps, can use the antibody with enzymatic activity, into free active medicine (referring to for example Massey, Nature328:457-458 (1987)), also referred to as " abzyme (abzymes) ", is changed prodrug of the present invention in this area.Can Dispersal risk-enzyme conjugates as described herein, for abzyme is delivered to tumor cell group.

Can be by technology well-known in the art by enzyme and antibody covalent bond, such as using the isodigeranyl functional cross-link agent.Perhaps, can use recombinant DNA technology well-known in the art to build the fusion rotein (referring to for example Neuberger et al., Nature312:604-608 (1984)) of at least antigen binding domain that comprises the antibody of the present invention partly be connected with at least functional activity of enzyme of the present invention.

Imagined more antibody modification.For example, can for example, by antibody and a kind of connection in multiple nonprotein character polymer, the copolymer of Polyethylene Glycol, polypropylene glycol, polyoxyalkylene or Polyethylene Glycol and polypropylene glycol.For example for example also the antibody bag can be stated from, for example, by (being respectively hydroxy methocel or gelatin microcapsule and poly-(methyl methacrylate) microcapsule) in condensation technique or the microcapsule for preparing by interfacial polymerization, in gluey drug delivery system (liposome, albumin microsphere, microemulsion, nano-particle and Nano capsule) or in macro emulsion.This type of technology is disclosed in " Remington ' sPharmaceutical Sciences ", and the 16th edition, Osol, A. compiles, and 1980.In order to extend the serum half-life of antibody, can, as for example United States Patent (USP) the 5th, will remedy receptor binding domain described in 739, No. 277 and mix antibody (especially antibody fragment).For this paper the time, IgG molecule (IgG for example " remedied receptor binding domain " and refer in term ₁, IgG ₂, IgG ₃or IgG ₄) the Fc district in be responsible for to extend the epi-position that IgG divides serum half-life in daughter.

Anti-DR5 antibody disclosed herein also can be mixed with immunoliposome.The method that can know by this area prepares the liposome that comprises antibody, such as Epstein et al., and Proc.Natl.Acad.Sci.USA82:3688 (1985); Hwang et al., Proc.Natl.Acad.Sci.USA77:4030 (1980); United States Patent (USP) the 4th, 485, No. 045 and the 4th, put down in writing in 544, No. 545.United States Patent (USP) the 5th, disclose the liposome that circulation time extends in 013, No. 556.Useful especially liposome can generate by reverse phase evaporation with the lipid composition that contains phosphatidylcholine, cholesterol and PEG derivatization phospholipid acyl ethanolamine (PEG-PE).Liposome is pushed through and has the filter that limits aperture, the liposome that has desired diameter with generation.Can put down in writing in J.Biol.Chem.257:286-288 (1982) as Martin et al., the Fab ' fragment of antibody of the present invention is reacted and the liposome coupling through disulfide exchange.Optionally in liposome, comprise chemotherapeutics (such as doxorubicin).Referring to Gabizon et al., J.National Cancer Inst.81 (19): 1484 (1989).

Antibody of the present invention comprises " crosslinked " DR5 antibody.Term " crosslinked " refers to that for this paper the time at least two IgG molecules combine to form (or single) molecule.DR5 antibody can be crosslinked with multiple connection molecule, and preferably, DR5 antibody is to use anti-IgG molecule, complement, chemical modification or molecular engineering and crosslinked.Once those skilled in the art will be appreciated that the antibodies cell surface membrane, complement antagonist molecule has relatively high affinity.Therefore, think that complement can be used as corsslinking molecular for connecting two or more Anti-DR5 antibodies of cell surface membrane institute combination.

5. recombination method

The present invention also provides the isolating nucleic acid of the DR5 antibody described herein of encoding, the carrier that comprises described nucleic acid and host cell, has reached for generating the recombinant technique of described antibody.

For the generation antibody of recombinating, separate its nucleic acid of coding, and insert replicable vector, for further cloning (DNA cloning) or expressing.Can use old process easily to separate DNA the order-checking (for example using the oligonucleotide probe that can be combined with the gene specific of encoding antibody) of encoding antibody.Can obtain many carriers.Support element generally include but be not limited to following one or more: signal sequence, origin of replication, one or more marker gene, enhancer element, promoter and transcription terminator.

Method herein comprises the method for the Anti-DR5 antibody for generating chimeric and restructuring, comprises the steps: to provide the carrier of the DNA sequence that comprises coding Anti-DR5 antibody light chain or heavy chain (or light chain and heavy chain the two); With described carrier transfection or transformed host cell; And cultivate described host cell under the condition that is enough to generation restructuring Anti-DR5 antibody product.

(i) signal sequence member

Anti-DR5 antibody of the present invention is direct recombinant production not only, and can be used as the fused polypeptide with heterologous polypeptide, and described heterologous polypeptide preferably has signal sequence or other polypeptide of specificity cleavage site in the N-end of mature protein or polypeptide.Selected allos signal sequence preferably is subject to host cell and identifies and process (be subject to signal peptidase cutting).Prokaryotic host cell for nonrecognition and processing natural antibody signal sequence, replace described signal sequence with the prokaryotic signal sequence that for example is selected from alkali phosphatase, penicillinase, lpp or thermally-stabilised enterotoxin 1 I targeting sequencing.For yeast secretary, can replace the natural signals sequence with for example signal described in yeast invertase targeting sequencing, alpha factor targeting sequencing (comprising saccharomyces and Crewe Vickers Saccharomyces alpha factor targeting sequencing) or acid phosphatase targeting sequencing, Candida albicans glucoamylase targeting sequencing or WO90/13646.In mammalian cell expression, can utilize mammalian signal sequence and viral secretory targeting sequencing, for example herpes simplex gD signal.

The DNA of this type of prosoma is connected in the reading frame of DNA of encoding antibody.

(ii) origin of replication member

The cloning and expression carrier all comprises the nucleotide sequence that can make carrier copy in the host cell of one or more selections.In general, in cloning vehicle, this sequence, for making carrier not rely on the sequence that host chromosome DNA copies, comprises origin of replication or autonomous replication sequence.This type of sequence of well-known various bacteria, yeast and virus.Origin of replication from plasmid pBR322 is suitable for most of gram negative bacteria, 2 μ plasmid starting points are suitable for yeast, and various viral starting point (SV40, polyoma virus, adenovirus, VSV or BPV) can be used for the cloning vehicle in mammalian cell.Usually, mammalian expression vector does not need origin of replication member (the SV40 starting point may only just be used because containing early promoter usually).

(iii) Select gene member

The cloning and expression carrier can comprise Select gene, also referred to as selection marker.The typical Select gene following protein of encoding: (a) give antibiotic or other toxin resistance, for example ampicillin, neomycin, methotrexate or tetracycline; (b) supply auxotrophy; Or (c) provide the crucial nutrient that can not be obtained by complex medium, the gene of the bacillus cereus D-alanine racemase of for example encoding.

An example of selection scheme utilizes medicine to block the growth of host cell.Those Hemapoiesis that successfully transform with heterologous gene are given the protein of drug resistance, thereby survive selection scheme.The example of this type of dominant selection is used medicine neomycin, mycophenolic acid and hygromycin.

Another example that is suitable for the selection marker of mammalian cell is the selection marker that can identify the cell of the picked-up antibody nucleic acid of having the ability, such as DHFR, thymidine kinase, metallothionein-I and-the preferred primates metallothionein gene of II, ADA Adenosine deaminase, ODC Ornithine decarboxylase etc.

For example, at first by all transformants are being contained to methotrexate (Mtx), a kind of competitive antagonist of DHFR, culture medium in cultivated to identify the cell transformed through the DHFR Select gene.When adopting wild type DHFR, suitable host cell is Chinese hamster ovary (CHO) cell line of the active defect of DHFR.

Perhaps, can by contain for selective agent such as the aminoglycoside antibiotics of selection marker for example in the culture medium of kanamycin, neomycin or G418 cultured cell select encoded Anti-DR5 antibody, wild type dhfr protein matter and another kind of selection marker such as aminoglycoside 3 '-DNA sequence of phosphotransferase (APH) transforms or the host cell (the wild type host who particularly comprises endogenous DHFR) of cotransformation.Consult United States Patent (USP) 4,965,199.

The Select gene that is applicable to yeast be present in trp1 gene in yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature282:39).The trp1 gene is for lacking the yeast mutant of energy for growth in tryptophan, and for example ATCC No.44076 or PEP4-1 provide selection marker.Jones exists trp1 infringement to provide for by not existing growth under tryptophan to detect the effective environment of conversion thereupon in 1977, Genetics 85:12. yeast host cell genome.Similarly, supply Leu2 defective yeast bacterial strain (ATCC20,622 or 38,626) with the known plasmid that carries the Leu2 gene.

In addition, the carrier derived from 1.6 μ m cyclic plasmid pKD1 can be used for transforming Crewe Vickers Saccharomyces cerevisiae.Perhaps, reported for the expression system at Kluyveromyces Lactis Vickers yeast large-scale production restructuring calf chymosin.Van den Berg，1990，Bio/Technology8：135。Also disclosed and be applicable to secrete the albuminised stable multicopy expression vector of ripe recombinant human serum by the industrial strain of Crewe Vickers Saccharomyces.Fleer et al.，1991，Bio/Technology9：968-975。

(iv) promoter member

The cloning and expression carrier comprises the promoter that is subject to host organisms identification usually, and it and antibody nucleic acid are operatively connected.The promoter that is applicable to prokaryotic hosts comprises that PhoA promoter, beta-lactamase and Lac operon system, alkali phosphatase, tryptophan (trp) promoter systems and hybrid promoter are such as the tac promoter.Yet other known antibacterial promoter is also suitable.Also will comprise for the promoter of bacterial system Shine-Dalgarno (S.D.) sequence that the DNA with the coding Anti-DR5 antibody is operatively connected.

Known eukaryotic promoter sequence.In fact, all eukaryotic genes all have the AT of being rich in district, are positioned at about 25 to 30 base places, initial upstream, site of transcribing.In the another kind of sequence of being permitted to find at base place, 70 to 80 of polygenic transcriptional start point upstreams, be the CNCAAT district, wherein N can be any nucleotide.At 3 of most of eukaryotic genes ' end, be the AATAAA sequence, it may be to add the signal of poly A tail to 3 of coded sequence ' end.In the suitable insertion carrier for expression of eukaryon of all these sequences.

The example that is applicable to the promoter sequence of yeast host comprises the promoter of glycerol 3-phosphate acid kinase or other glycolytic ferment, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, GPI, 3-phoshoglyceric acid mutase, pyruvate kinase, phosphotriose isomerase, glucose phosphate isomerase and glucokinase.

As thering is other Yeast promoter of controlling the inducible promoter of the additional advantage transcribe by growth conditions, be alcoholdehydrogenase 2, different cell pigment C, acid phosphatase, the digestive enzyme relevant with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and the promoter region of the enzyme of responsible maltose and galactose utilization.The carrier and the promoter that are applicable to yeast expression are further described in EP73,657.The yeast enhancer also can be favourable with the Yeast promoter coupling.

Transcribing Anti-DR5 antibody by carrier in mammalian host cell for example is subject to by virus such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferred simian virus 40 (SV40) genome, by for example actin promoter or immunoglobulin promoter, and the control of the promoter that obtained by the heat shock promoter of allos mammalian promoter, if the compatible words of this type of promoter and host cell systems.

Obtain easily the early stage and late promoter of SV40 virus with the form of SV40 restriction fragment, this fragment also comprises SV40 virus replication starting point.Obtain easily the immediate early promoter of human cytomegalic inclusion disease virus with the form of HindIII E restriction fragment.United States Patent (USP) 4,419, disclose the system of bovine papilloma virus as carrier expressible dna in mammalian hosts of using in 446.United States Patent (USP) 4,601, described a kind of improvement of this system in 978.Also can consult Reyes et al., 1982, Nature297:598-601 about the control following table intelligent beta-interferon cDNA at the thymidine kinase promoter from herpes simplex virus in mouse cell.Perhaps, can use the rous sarcoma virus long terminal repeat as promoter.

(v) enhancer element member

Usually by insert enhancer sequence in carrier, improve higher eucaryotic cells transcribing the DNA of code book invention Anti-DR5 antibody.Known many enhancer sequence from mammalian genes (globulin, elastoser, albumin, α-fetoprotein and insulin).Yet, usually use the enhancer from eukaryotic cell virus.Example comprises enhancer (bp100-270), the sub-enhancer of cytomegalovirus early promoter of SV40 origin of replication side in late period one, enhancer and the adenovirus enhancer of polyoma virus origin of replication side in late period one.Also can consult Yaniv, 1982, Nature297:17-18 about the enhancing element that activates eukaryotic promoter.Enhancer can montage in carrier, be positioned at 5 of antibody coding sequence ' or 3 ' position, but be preferably placed at 5 ' site of promoter.

(vi) tanscription termination member

Also will comprise and stop transcribing and the necessary sequence of stable mRNA for the expression vector of eukaryotic host cell (yeast, fungus, insecticide, plant, animal, people or from the nucleated cell of other multicellular organisms).This type of sequence can be obtained by 5 of eucaryon or viral DNA or cDNA untranslated region ' end and 3 ' end once in a while usually.These district inclusions become the nucleotide section of polyadenylation fragment at the untranslated part transcription of the mRNA of coding multivalent antibody.A kind of useful tanscription termination member is bovine growth hormone polyadenylation district.Consult WO94/11026 and reach wherein disclosed expression vector.

(vii) selection of host cell and conversion

The host cell that is suitable for the DNA in clone or expression this paper carrier is above-described prokaryote, yeast or higher eucaryotic cells.The prokaryote that is suitable for this purpose comprises eubacteria, such as Gram-negative or gram-positive organism, enterobacteriaceae for example, for example, such as Escherichia (Escherichia) colon bacillus (E.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonella (Salmonella) is Salmonella typhimurium (Salmonella typhimurium) for example, Serratia (Serratia) is serratia marcescens (Serratia marcescans) for example, and Shigella (Shigella), and bacillus (Bacilli) for example, such as bacillus subtilis (B.subtilis) and Bacillus licheniformis (the B.licheniformis) (DD266 published on April 12nd, 1989, disclosed Bacillus licheniformis 41P in 710), Rhodopseudomonas (Pseudomonas) is such as Pseudomonas aeruginosa (P.aeruginosa), and streptomyces (Streptomyces).A kind of preferred escherichia coli cloning host is escherichia coli 294 (ATCC31,446), although other bacterial strain such as escherichia coli B, escherichia coli X1776 (ATCC31,537) and escherichia coli W3110 (ATCC27,325) are also suitable.These examples are illustration and unrestricted.

Except prokaryote, eukaryotic microorganisms, be also suitable clone or the expressive host of the carrier of encoding D R5 antibody such as filamentous fungi or yeast.Wine brewing sugar yeast (Saccharomyces cerevisiae) or bakery yeast commonly used are the most frequently used eucaryon host microorganisms such as low.Yet, usually can obtain many other genus and species and bacterial strain and can be used for the present invention, such as foxtail millet wine fragmentation sugar yeast (Schizosaccharomyces pombe); Kluyveromyces (Kluyveromyces) host such as for example Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis) (ATCC12,424), Bulgarian kluyveromyces (K.bulgaricus) (ATCC16,045), Brunswick kluyveromyces (K.wickeramii) (ATCC24,178), K.waltii (ATCC56,500), fruit bat kluyveromyces (K.drosophilarum) (ATCC36,906), heat-resisting kluyveromyces (K.thermotolerans) and marxianus yeast (K.marxianus); Inferior sieve Saccharomyces (Yarrowia) (EP402,226); Pichia pastoris phaff (Pichia pastoris) (EP183,070); Mycocandida (Candida); Rui Shi Trichoderma spp. (Trichoderma reesia) (EP244,234); Neuraspora crassa (Neurospora crassa); Permitted all so prosperous yeast of prosperous Saccharomyces (Schwanniomyces) (Schwanniomyces occidentalis); With filamentous fungi such as for example Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (Tolypocladium) and aspergillus (Aspergillus) host such as aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Be applicable to express the host cell of glycosylated antibodies derived from multicellular organisms.The example of invertebral zooblast comprises plant and insect cell.Identified many baculovirus strains and variant and the corresponding insect host cell that allows, they are from hosts such as fall army worm Spodoptera frugiperda (caterpillar), Aedes aegypti Aedes aegypti (mosquito), Aedes albopictus Aedes albopictus (mosquito), Drosophila melanogaster Drosophila melanogaster (fruit bat) and silkworm Bombyx mori.The public can obtain the Various Diseases strain for transfection, the for example Bm-5 strain of the L-1 variant of autographa california Autographa californica NPV and BmSNPV, and this viroid can be according to the present invention as the virus of this paper, especially for transfection fall army worm cell.

Also can utilize the plant cell cultures of cotton, Semen Maydis, Rhizoma Solani tuber osi, Semen sojae atricolor, petunia, Fructus Lycopersici esculenti and Nicotiana tabacum L. as the host.

Then, vertebrate cells obtains extensive concern, and in cultivation (tissue culture), the breeding of vertebrate cells has become old process.The example of useful mammalian host cell line is the monkey kidney CV1 system (COS-7, ATCCC RL1651) transformed with SV40; Human embryo kidney (HEK) system (293 or for growth in suspension culture 293 cells of sub-clone, Graham et al., 1977, J.Gen Virol.36:59); Baby hamster kidney cell (BHK, ATCC CCL10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al., 1980, Proc.Natl.Acad.Sci.USA77:4216)); Mice Sai Tuoli (sertoli) cell (TM4, Mather, 1980, Biol.Reprod.23:243-251); Monkey-kidney cells (CV1, ATCCCCL70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); Human cervical carcinoma cell (HELA, ATCC CCL2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL34); Cattle Mus (buffalorat) hepatocyte (BRL 3A, ATCC CRL1442); Human pneumonocyte (W138, ATCC CCL75); Human liver cell (Hep G2, HB8065); Mouse mammary tumor (MMT060562, ATCC CCL51); TRI cell (Mather et al., 1982, Annals N.Y.Acad.Sci.383:44-68); The MRC5 cell; The FS4 cell; People's hepatoma system (Hep G2); For example, with myeloma or lymphoma cell (Y0, J558L, P3 and NS0 cell) (referring to United States Patent (USP) 5,807,715).

Host cell is transformed with the above-mentioned expression generated for antibody or cloning vehicle, and cultivate in the conventional Nutrient medium of the gene for evoked promoter, selection transformant or amplification coding expectation sequence and appropriate improvement.

(viii) cultivate host cell

Can in multiple culture medium, cultivate for generating the host cell of antibody of the present invention.The EagleShi culture medium (DMEM, Sigma) of commercially available culture medium such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), RPMI-1640 (Sigma) and DulbeccoShi improvement is suitable for cultivating host cell.In addition, can use in following document any culture medium of describing culture medium as host cell: Ham et al., 1979, Meth.Enz.58:44; Barnes et al., 1980, Anal.Biochem.102:255; United States Patent (USP) 4,767,704; 4,657,866; 4,927,762; 4,560,655; Or 5,122,469; WO90/03430; WO87/00195; Or United States Patent (USP) reexamination 30,985.Any these culture medium as required supplementing hormone and/or other somatomedin (such as insulin, transferrin or epidermal growth factor), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer agent (such as HEPES), nucleotide (such as adenosine and thymidine), antibiotic (such as GENTAMYCIN ^tMmedicine), trace element (being defined as the common inorganic compound existed with the final concentration of micro-molar range) and glucose or the equivalent energy.Can also with debita spissitudo comprise those skilled in the art will know that any other must fill-in.Before condition of culture such as temperature, pH etc. select for host cell for expression, and this is obvious for those of ordinary skill.

(ix) purification

When using recombinant technique, can in cell, in periplasmic space, generate antibody, or direct secretion is in culture medium.If generate antibody in cell, so as the first step, remove the microgranule fragment of host cell or crack fragment by for example centrifugal or ultrafiltration.Carter et al., 1992, Bio/Technology10:163-167 has described the flow process for separating of the antibody that is secreted into the colibacillus periplasm space.Briefly, when having sodium acetate (pH3.5), EDTA and Phenylmethanesulfonyl fluoride (PMSF), make cell stick with paste and melt approximately 30 minutes.Can be by the centrifugal cell debris of removing.If by antibody-secreting in culture medium, commodity in use protein compression filter at first, for example concentrated supernatant from this type of expression system of AMICON or MILLIPORE PELLICON ultra filtration unit so usually.In any above-mentioned steps, can comprise that protease inhibitor such as PMSF carrys out the Profilin hydrolysis, and can comprise that antibiosis usually prevents the growth of external contaminant.

Can use the antibody compositions that for example hydroxyapatite, gel electrophoresis, dialysis and affinity chromatograph come purification to be prepared by cell, preferred purification technique is affinity chromatograph.Protein A depends on kind and the isotype of any immunoglobulin fc region existed in antibody as the suitability of affinity ligand.Protein A can be used for the antibody of purification based on people γ 1, γ 2 or γ 4 heavy chains (Lindmark et al., 1983, J.Immunol.Meth.62:1-13).Protein G is recommended for all mice isotypes and people γ 3 (Guss et al., 1986, EMBO J.5:1567-1575).What the accompanying substrate of affinity ligand was the most frequently used is agarose, but can use other substrate.The substrate of physically stable such as controllable bore diameter glass or poly-(styrene divinyl) benzene can obtain than agarose flow velocity and shorter process time faster.For comprising C _hthe antibody of 3 domains, can be used Bakerbond ABX ^tMresin (J.T.Baker, Phillipsburg, NJ) carries out purification.According to antibody to be recycled, also can use chromatography, heparin SEPHAROSE on other oroteins purification technique such as the fractional distillation on ion exchange column, ethanol precipitation, reversed-phase HPLC, tripoli ^tMon chromatography, anion or cation exchange resin (such as the poly-aspartate post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

the purposes of B.DR5 antibody

DR5 antibody of the present invention has multiple effectiveness.

Known DR5 mediated apoptosis signal.Although the normal cell of a few types is expressed DR5, but as if mainly be confined to tumor cell through the apoptotic signal of receptor thus, they are being subject under the background that oncogene such as MYC or RAS transform becoming to the apoptosis of death receptor mediation susceptible (Wang et al., Cancer Cell 5:501-12 (2004) more; Nesterov et al., Cancer Res.64:3922-7 (2004)).DR5 is expressed by human carcinoma cell line and primary tumor frequently.So, Anti-DR5 antibody can be used for diagnosis and the treatment of cancer.For example, the DR5 agonistic antibody is used in the method that mammal comprises treatment cancer in the people.In these methods, the independent or DR5 antibody that combine other therapeutic agent or technology to administration, preferably agonistic antibody.Described cancer can be the cancer of the expression DR5 of any type, comprises solid tumor, and particularly when accepting existing therapy, tumor is more made progress (progression) or be there is no late period or a metastatic solid tumors of effective known therapies.The particular type of cancer includes but not limited to colorectal carcinoma, nonsmall-cell lung cancer (NSCLC), cancer of pancreas, ovarian cancer, breast carcinoma, non-Hodgkin's (Hodgkin) lymphoma (NHL), glioblastoma or melanoma, preferably colorectal carcinoma, NSCLC or NHL.

In addition, DR5 antibody can be used for diagnosis and the treatment that mammal comprises other DR5 related diseases illness of science in the people, such as immune correlated disease.

In mammal, the diagnosis of various pathology illness described herein can be undertaken by skilled practitioner.This area can obtain for example being diagnosed or detect cancer in mammal or the diagnostic techniques of immune correlated disease.For example, cancer can be identified by following technology, includes but not limited to palpation, hemanalysis, X ray, NMR, like that.

Immune correlated disease also can be easy to identify.

In systemic lupus erythematosus (sle), the main medium of disease is that the autoreactivity antibody generated oneself protein matter/tissue reaches the immune-mediated inflammation occurred subsequently.A plurality of organs and system are affected clinically, comprise kidney, lung, musculoskeletal system, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood.

Rheumatoid arthritis (RA) is a kind of chronic systemic autoimmunity inflammatory diseases, mainly involves the synovial membrane in a plurality of joints, causes thus the damage to articular cartilage.Pathogenesis relies on the T lymphocyte, and and rheumatoid factor, relevant for the generation of the autoantibody of Autologous IgG, cause thus the formation of immune complex, it reaches high level in joint fluid and blood.These complex in joint can bring out significant lymphocyte and monocyte infiltration reaches significant synovial membrane variation subsequently in synovial membrane; Articular cavity/liquid is subject to the cytoid infiltration of class and adds many neutrophil cells.Affected tissue is mainly joint, is usually symmetrical pattern.Yet outside joint, also there are two kinds of principal modes in disease.A kind of form is to form outside joint to damage, with the typical damage of ongoing gradual arthrosis and pulmonary fibrosis, vasculitis and skin ulcer.Outside joint, the second form of disease is so-called Fil Ti Shi (Felty) syndrome, and it occurs in the RA late cases, sometimes in arthrosis after silence, and relates to and occurs that neutrophil cell reduces disease, thrombocytopenia and splenomegaly.This can be accompanied by vasculitis and infraction, skin ulcer and gangrenous formation in a plurality of organs.The patient also usually forms rheumatoid nodule in the subcutaneous tissue on influenced joint; The tuberosity in late period has the downright bad center surrounded by the inflammatory cell infiltration thing mixed.Other performance that can occur in RA comprises: pericarditis, pleuritis, coronaritis, the interstitial pneumonia with pulmonary fibrosis, keratoconjunctivitis sicca and rheumatoid nodule.

Juvenile chronic arthritis is a kind of chronic idiopathic inflammatory diseases, usually starts from before 16 years old.Its phenotype is a bit similar to RA; Some patients of the rheumatoid factor positive are included into juvenile rheumatoid arthritis.Disease is subdivided into three kinds of primary categories: the minority joint, multiarticulate and systematic.Arthritis can be serious, normally destructive, and causes ankylosis and growth retardation.Other performance can comprise chronic anterior uveitis and systemic amyloidosis.

Spondyloarthropathy is to have some general Clinical symptoms and the general one group disease relevant with the HLA-B27 gene product expression.These diseases comprise: ankylosing spondylitis, Lai Teershi (Reiter) syndrome (reactive arthritis), the arthritis relevant with inflammatory bowel, the spondylitis relevant with psoriasis, young hair style spondyloarthropathy and indifference (undifferentiated) spondyloarthropathy.Distinctive feature comprises having or do not have spondylitic sacroiliitis; The asymmetric arthritis of inflammatory; Relevant with HLA-B27 (allele of the MHC I type HLA-B locus defined in serology); The inflammation of eye; Reach the not autoantibody relevant with other atrophic diseases.For inducing an illness, crucial relevant cell is the CD8+T lymphocyte, and targeting is by the cell of the antigen of MHC I type molecular presentation.The CD8+T cell can react with MHC I type allele HLA-B27, just as it is the exogenous peptide by MHC I type developed by molecule.Existing hypothesis, the epi-position of HLA-B27 may be simulated the antigenic epitopes of antibacterial or other microorganism, therefore induces the CD8+T cell response.

Etiology the unknown of systemic sclerosis (scleroderma).The characteristics of disease are skin sclerosis; This may be induced by active inflammatory process.Scleroderma can be part or systematic; Blood vessel injury is general, and the endothelial cell damage in Microvasculature is the developing early stage and important event of systemic sclerosis; Blood vessel injury may be by immune-mediated.There is the monocyte infiltration thing in skin injury and have antinuclear antibody hint amynologic basis in many patients.ICAM-1 usually in skin injury fibroblastic cell surface raise, illustrate that T cell and these cell interactions may work in the pathogeny of disease.Other organ involved comprises: gastrointestinal tract: smooth muscle atrophy and fibrosis cause abnormal wriggling/motion; Kidney: concentricity interior subcutaneous neointimal hyperplasia (concentric subendothelial intimalproliferation), affect little arc and interlobar arteries, thereby reduce the renal cortex blood flow, cause albuminuria, azotemia and hypertension; Skeletal muscle: atrophy, interstitial fibrosis, inflammation; Lung: interstitial pneumonia and interstitial fibrosis; And the heart: contraction bands is downright bad, cicatrix/fibrosis.

The idiopathic inflammatory myopathy, comprise dermatomyositis, polymyositis and other, is the chronic muscle inflammatory disease that causes the unknown etiology of muscle weakness.Muscle injury/inflammation is usually symmetrical with progressive.Autoantibody is relevant with most of forms.These myositis specific autoantibodies for and suppress to relate to the function of composition, protein and the RNA of protein synthesis.

Si Yegelunshi (Sj

gren) syndrome is by immune-mediated inflammation and lachrymal gland and salivary gland function are destroyed and caused subsequently.This disease may be relevant with the inflammatory connective tissue disease or be accompanied by the inflammatory connective tissue disease.This disease is relevant with the autoantibody generation for Ro and La antigen, and these two kinds of antigens are all little RNA-protein complexes.Infringement causes keratoconjunctivitis sicca, dry mouth, reaches other performance or association, comprises biliary cirrhosis, periphery or esthesioneurosis and palpable purpura.

Systemic vasculitis comprises such disease, and wherein primary injury is inflammation, and the follow-up damage to blood vessel causes by the tissue ischemia/necrosis of influenced vascularity/degeneration, and finally causes the terminal organ dysfunction in some situation.Secondary injury or sequela that vasculitis also can be used as other immunity-inflammation mediated disease such as rheumatoid arthritis, systemic sclerosis etc. occur, and particularly also with immune complex, are forming in relevant disease.Disease in primary systemic vasculitis group comprises: systemic necrotizing vasculitis: polyarteritis nodosa, allergic angiitis and granulomatosis, polyangitis; Wei Genashi (Wegener) granulomatosis; Lymphomatoid granulomatosis; And giant cell arteritis.Various vasculitises comprise: mucosa and skin lymph node syndrome (MLNS or river Ji Shi (Kawasaki) disease), the CNS vasculitis separated, Bei Qieteshi (Behet) disease, thromboangiitis obliterans (Bai Geshi (Buerger) disease) and cutaneous necrosis trichodophlebitis.The vasculitic pathogeny of listed most of types thinks it is mainly to deposit in blood vessel wall due to immunoglobulin complex and with by ADCC, complement activation or inflammatory response that both induce.

Sarcoidosis (sarcoidosis) is etiology the unknown, is characterised in that the illness that all has epithelioid granuloma in any tissue of body almost; Modal is to involve lung.Pathogeny relates to the macrophage of disease sites sustainable existence activation and lymphoid cell and discharges local and systemic activated product by these cell types subsequently and the chronic sequela that causes.

Autoimmune hemolytic anemia, comprise autoimmune hemolytic anemia, immunopancytopenia and paroxysmal nocturnal hemoglobinuria, owing to generating with erythrocyte (with in some situation and other hemocyte, comprise platelet) result of the antigen reactive antibody of surface expression is the reflection of through dissolving and/or the receptor-mediated mechanism of ADCC/Fc of complement-mediated, removing those antibody-coated cellses.

At AT, comprise thrombocytopenic purpura, in thrombocytopenia immune-mediated in other clinical settings, due to antibody or complement is attached to platelet and with by the receptor-mediated mechanism of complement dissolving, ADCC or Fc, removing platelet destruction/removal occurs.

Thyroiditis, comprise Ge Leifusishi (Graves) disease, Hashimoto disease (Hashimoto) thyroiditis, teenager lymphocytic thyroiditis and atrophic thyroiditis, be due to the autoimmune response for thyroid antigen and produce and be present in thyroid and the antibody of special proteins react to thyroid usually.Existing experimental model comprises spontaneous model: rat (BUF and BioBreeding rat) and chicken (fat chicken strain); But guidance model: with Elityran, thyroid microsomal antigen (thyroid peroxidase) immune animal.

Type i diabetes or insulin-dependent diabetes are that the autoimmunity of beta Cell of islet is destroyed; This destroys by autoantibody and autoreactive T cell mediation.The antibody of insulin or Insulin receptor INSR also can produce the phenotype that insulin does not respond.

Immune-mediated nephropathy, comprise glomerulonephritis and tubulointerstitial nephritis, it is the damage to nephridial tissue due to antibody or T cell mediated, or directly due to the autoreactivity antibody or the T cell that generate for kidney antigen, or indirectly because antibody and/or immune complex for other non-responding property of kidney antigen deposit in kidney.Therefore, other the immune-mediated disease that causes immune complex to form also can be used as the nephropathy of indirect sequelae induction of immunity mediation.Directly and indirectly immunologic mechanism all causes inflammatory response, and it produces/induce damage and forms in nephridial tissue, causes organ dysfunction impaired, in some situation, develops into renal failure.Two kinds of immunologic mechanisms of body fluid and cell all can relate to the pathogeny of damage.

The demyelinating disease of maincenter and peripheral nervous system, comprise multiple sclerosis; Idiopathic demyelination polyneuropathy or Ge-Ba Er Shi (Guillain-Barr é) syndrome; With chronic inflammatory demyelination polyneuropathy, think and there is the autoimmune basis, and owing to oligodendrocyte or to marrow squama fat, directly causing damage and causing nerve demyelination.In MS, evidence suggests inducing and develop and relying on the T lymphocyte of this disease.Multiple sclerosis is to rely on the lymphocytic demyelinating disease of T, and has recurrence-alleviation process or very long progressive process.Cause of disease the unknown; Yet viral infection, hereditary cachexia, environment and autoimmunity all can work.The macrophage that damage contains main infiltrate, microglia and infiltration by the T cell mediated; The CD4+T lymphocyte is the main cell type of injury region.Oligodendrocyte death and mechanism the unknown of demyelination subsequently, but may be driven by the T lymphocyte.

Inflammatory and fibrosis pneumonopathy, comprise the eosinocyte pneumonia; Idiopathic pulmonary fibrosis and hypersensitivity pneumonia, may relate to not modulated immunity-inflammation and reply.Suppressing this replys and will have the treatment benefit.

Autoimmune or immune-mediated dermatosis, comprise bullous dermatosis, erythema multiforme and contact dermatitis, and by the autoantibody mediation, T lymphocyte occurs to rely on for it.

Psoriasis is the inflammatory diseases of T cell mediated.Damage contains T lymphocyte, macrophage and antigen processing cell, and the infiltrate of some neutrophil cells.

Allergic disease, comprise asthma; Allergic rhinitis; Atopic dermatitis; The food hypersensitivity; And urticaria, they are that the T lymphocyte relies on.The inflammation of the inflammation that these diseases are mainly induced by the T lymphocyte, IgE mediation or the two combination mediation.

Transplant relevant disease, comprise transplant rejection and graft versus host disease (GVHD), rely on the T lymphocyte; Suppress the T lymphocyte function and there is the improvement effect.

Wherein by intervening the Other diseases that immunity and/or inflammatory response are useful, infectious disease is arranged, include but not limited to viral infection (including but not limited to AIDS, A type, B-mode, the third type, fourth type, hepatitis E), antibacterial infection, fungal infection, and protozoacide and parasitic infection (stimulate the molecule (or derivant/agonist) of MLR can be for strengthening the immunne response to infectious agent in treatment); Immunodeficiency (stimulate molecule/derivant of MLR/agonist can be in treatment for strengthening the immunne response to the illness of (as infected at HIV) or iatrogenic (as be derived from chemotherapy) immunodeficiency of heritability, acquired, infection-induced); And neoplasia.

Antibody, preferably at carrier, preferably is applied to mammal in pharmaceutical acceptable carrier.Suitable carrier and preparaton thereof is recorded in " Remington ' s Pharmaceutical Sciences ", and the 16th edition, 1980, Mack Publishing Co., the people such as Oslo compile.Be typically, in preparaton, use the pharmaceutically acceptable salt of Sq to make preparaton etc. ooze.The example of carrier comprises saline, woods Ge Shi (Ringer) solution and dextrose solution.The pH of solution preferably approximately 5 to approximately 8, and more preferably from about 7 to approximately 7.5.Other carrier comprises extended release preparation, and such as the solid hydrophobic polymer semi permeability substrate that contains antibody, this substrate is the form of approved product, for example thin film, liposome or microcapsule.It will be apparent for a person skilled in the art that some carrier may be preferred according to for example using the concentration of path and institute's administration of antibodies.

Antibody can for example, be applied to mammal by injection (intravenous, intraperitoneal, subcutaneous, intramuscular, intraportal), or by guaranteeing to be delivered to other method of blood flow such as perfusion with effective form.Antibody also can be used by isolated organ perfusion (isolated perfusion) technology, such as the separating tissues perfusion, with performance topical therapeutic effect.Part or intravenous injection are preferred.

The effective dose of administration of antibodies and timetable can be determined by rule of thumb, make this type of and determine in the art technology scope.It will be understood by those skilled in the art that the antibody dosage that must use will depend on the mammal that for example will accept antibody, and use the particular type of path, antibody used and be applied to mammiferous other medicines.Select the guidance of antibody optimal dose to see the document about the Antybody therapy purposes, for example " Handbook of Monoclonal Antibodies ", the people such as Ferrone compile, Noges Publications, Park Ridge, NJ, 1985, the 22 chapters, the 303-357 page; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389.The typical daily dose that antibody is used separately can in every day, approximately 1 μ g/kg body weight be to the scope of 100mg/kg body weight nearly or more, and this depends on above-mentioned factor.

Antibody can be co-administered in mammal with one or more other therapeutic agents of effective dose.In the treatment of cancer, especially true, because the deactivation of many tumors by the p53 tumor suppressor gene obtained the resistance to chemotherapy or radiotherapy.Because DR5 stimulates apoptosis in the mode that does not rely on p53, therefore expect that it not only can be used as clinically single medicament and uses, but also can combine the treatment of cancer of other type, such as for example chemotherapy (chemotherapeutics), radiotherapy, immunological adjuvant (immunoadjuvant), growth inhibitor, cytotoxic agent and/or cytokine.Also can adopt known other medicament apoptosis-induced in mammalian cell, this type of medicament comprises TNF-α, TNF-β, CD30 part, 4-1BB part and Apo2L/TRAIL, and other antibody that can be apoptosis-induced.Described one or more other therapies can comprise therapeutic antibodies (beyond DR5 antibody), and this antibody-like can comprise that anti-Her receptor antibody is (such as HERCEPTIN

(trastuzumab), Genentech, Inc.), VEGF antibody, anti-CD 20 antibodies be (such as RITUXAN (rituximab), Genentech, Inc.) and for the antibody of other receptor of Apo2L/TRAIL such as anti-DR4 antibody or for other TNF receptor family member's antibody such as ENBREL

(etanercept) (Immunex).

The contemplated chemotherapy of the present invention comprises known in the art and obtainable chemical substance or medicine, such as doxorubicin (Doxorubicin), 5-fluorouracil (5-Fluorouracil), etoposide (etoposide), camptothecine (camptothecin), folinic acid (Leucovorin), cytosine arabinoside (Cytosine arabinoside), cyclophosphamide (Cyclophosphamide), Tespamin (Thiotepa), busulfan (Busulfan), Cytoxin, taxol (Taxol), methotrexate (Methotrexate), cisplatin (Cisplatin), melphalan (Melphalan), vinblastine (Vinblastine) and carboplatin (Carboplatin).The preparation of this based chemotherapy and dosage administration time table can be used according to the description of manufacturer, or are determined by rule of thumb by skilled practitioner.The preparation of this based chemotherapy and dosage administration time table also are recorded in " Chemotherapy Service ", and M.C.Perry compiles, Williams& Wilkins, Baltimore, MD (1992).

Chemotherapy is preferably used in pharmaceutical acceptable carrier, as described above such as those.It is identical that the mode of administration of chemotherapy can adopt with DR5 antibody, or it can be applied to mammal through different mode.For example, but administering to a mammal DR5 antibody and Orally administered chemotherapy.

Can be according to this area commonly used scheme of knowing with those of skill in the art of radiotherapy is applied to mammal.This type of therapy can comprise caesium, iridium, iodine or cobalt irradiation.Radiotherapy can be total irradiation, or can be local for the specific part in health or on health or tissue.Be typically, radiotherapy is approximately being used with pulse mode for 1 one period of thoughtful approximately 2 weeks.Yet radiotherapy can be used in one period of longer time.Optional, radiotherapy can be used with single dose or continuous multi-agent.

Antibody can or be used with one or more other therapeutic agent orders simultaneously.The amount of antibody and therapeutic agent depends on for example type, the pathology illness for the treatment of and the timetable of using and the path of medicine used, but is usually less than the amount while using separately separately.

After administration antibody, can monitor mammiferous physiology's situation with the well-known various ways of skilled practitioner.

Imagine Antagonism or barrier DR5 antibody and can be used for treatment.For example, DR5 antibody can be applied to mammal (as described above all) with blocking-up DR5 receptors bind Apo-2L, improve thus the bioavailability of the Apo-2L that uses during the Apo-2L therapy with apoptosis-induced in cancerous cell.

The therapeutic effect of DR5 antibody of the present invention is checked with the interior animal model of use body in algoscopy in vitro.Multiple well-known animal model can be used for further understanding the DR5 antibody identified herein in the formation of for example cancer or immune correlated disease and the effect in pathogeny, and the effect of test candidate therapeutic agent.In the body of this class model, essence makes them can forecast the response in human patients especially.

The animal model of immune correlated disease comprises (genetically modified) two kinds of animals nonrecombinant and restructuring.Non-recombinant animal model comprises for example Rodents, for example mouse model.But this class model Application standard technology, for example the implantation under subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation and the scrotum, generate by cell being imported to homogenic mice.

For example the animal model of graft versus host disease is known.Graft versus host disease occurs in immunocompetent cell being transplanted to immunity containment or tolerance patient the time.Host antigen is identified and replied to donorcells.Reply and can change from life-threatening serious inflammation to the slight case of suffering from diarrhoea and losing weight.Graft-versus-host Disease Model provides the means of assessment for the t cell responses of MHC antigen and less important graft antigen.A kind of suitable flow process is recorded in " Current Protocols in Immunology ", unit 4.3 in detail.

The animal model of skin allograft rejection is the means that the cell-mediated in-vivo tissue of test T destroys, and its indicates and measure its effect in antiviral and tumour immunity.The most frequently used and generally acknowledged model is used Mus tail skin graft.Repeated experiments proves the skin allograft rejection by T cell, helper T cell and lethal effect T cell but not is antibody-mediated.Auchincloss, H.Jr.andSachs, D.H., " Fundamental Immunology ", the 2nd edition, W.E.Paul compiles, Raven Press, NY, 1989,889-992.A kind of suitable flow process is recorded in " Current Protocols inImmunology ", unit 4.4 in detail.Other transplant rejection model that can be used for testing the present composition has the allogene Heart Transplantation Model, is recorded in Tanabe, M.et al., Transplantation, 58:23 (1994) and Tinubu, S.A.et al., J.Immunol., 4330-4338 (1994).

The animal model of delayed hypersensitivity provides the algoscopy of cell-mediated immunologic function equally.The delayed hypersensitivity reaction is immunne response in the cell-mediated body of T, is characterized as the inflammation that just reaches peak value after antigen is attacked after a period of time passage.These reactions also occur in the tissue specificity autoimmune disease, such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a kind of model of MS).A kind of suitable flow process is recorded in " Current Protocols in Immunology ", unit 4.5 in detail.

Arthritic animal model is collagen-induced arthritis.This model is shared clinical, histology and the amynologic characteristic of human autoimmune rheumatoid arthritis, is the arthritic accepted model of human autoimmune.The erosion that is characterized as synovitis, cartilage and subchondral bone of Mouse and rat model.DR5 antibody of the present invention can be used " Current Protocols in Immunology ", sees above, and in unit 15.5, the scheme of record comes testing needle to the arthritic activity of autoimmunity.Also can be referring to using Issekutz, A.C.et al., Immunology, the model of the monoclonal antibody of the CD18 of record and VLA-4 integrin in 88:569 (1996).

Put down in writing a kind of asthmatic model, airway hyperreactivity, ensinophilosis and inflammation that wherein inducing antigen brings out by the same protein confrontation animal attack that makes the animal sensitization with ovalbumin, then use to deliver by aerosol.Several animal models (Cavia porcellus, rat, non-human primates) show the symptom similar to people's atopic asthma after attacking with aerosol antigen.Mouse model has many features of people's asthma.In treatment, the activity in asthma and the appropriate method of effect are recorded in Wolyniec, W.W.et al., Am.J.Respir.Cell Mol.Biol., 18:777 (1998) and the list of references of quoting thereof to the test present composition.

In addition, DR5 antibody of the present invention can be tested on the animal model of psoriasiform disease.DR5 antibody of the present invention can be at Schon, M.P.et al., and Nat.Med., test in the scid/scid mouse model of 3:183 (1997) record, and its small mouse is showed and the similar histopathology skin injury of psoriasis.Another kind of suitable model is as Nickoloff, B.J.et al., Am.J.Path., the application on human skin of the described preparation of 146:580 (1995)/scid mouse Chimera.

As everyone knows for the safety of testing the candidate therapeutic compositions and the several animal models of active anticancer.These animal models comprise the xenotransplantation of people's tumor in nude mouse and/or scid/scid mice, or hereditary Mus tumor model (genetic murine tumor model) is such as the p53 knock-out mice.

Restructuring (transgenic) animal model can be transformed by the genome of the coded portion importing purpose animal of the molecule by identifying herein by the standard technique for generating transgenic animal.The animal that can serve as the transgeneic procedure object includes but not limited to mice, rat, rabbit, Cavia porcellus, sheep, goat, pig and non-human primates, and for example baboon, chimpanzee and monkey, such as macaque.Known in the artly for the technology that transgenic is imported to this type of animal, comprise pronuclear microinjection (pronucleic microinjection) (Hoppeand Wanger, United States Patent (USP) the 4th, 873, No. 191); The gene transfer of retrovirus-mediated method enters germline (for example Van der Putten et al., Proc.Natl.Acad.Sci.USA, 82:6148-615 (1985)); Gene targeting in embryonic stem cell (Thompson et al., Cell, 56:313-321 (1989)); Embryo's electroporation (Lo, Mol.Cel.Biol., 3:1803-1814 (1983)); The gene transfer of Sperm-mediated (Lavitrano et al., Cell, 57:717-73 (1989)).Summary is referring to for example United States Patent (USP) the 4th, 736, No. 866.

For the purposes of the present invention, transgenic animal comprise that those carry genetically modified animal (" inlaying animal ") in its part cell.Transgenic can be used as single transgenic or for example head-head or the head-tail series connection thing integration of series connection thing.Also may be as follows the transgenic selectivity be imported to particular cell types, Laskoet al. for example, Proc.Natl.Acad.Sci.USA, the technology of 89:6232-636 (1992).

The expression of transgenic in transgenic animal can be monitored by standard technique.For example, Southern engram analysis or pcr amplification can be used for verifying genetically modified integration.Then can use the technical Analysis mrna expression level such as in situ hybridization, Northern engram analysis, PCR or immunocytochemistry.Animal can further be checked the pathological sign of immunological disease, for example by histological inspection, to measure immunocyte, enters the infiltration of particular organization or existing of carcinous or malignant tissue.

Perhaps, can build " knocking out " animal, it is due to the endogenous gene of the polypeptide identified herein of coding and import the homologous recombination between the genomic DNA of this same polypeptide of coding of change of animal embryo cell and tool is defective or the gene of this polypeptide of coding of changing.For example, encode the cDNA of specific polypeptide can be according to the technology of having set up the genomic DNA for this polypeptide of clones coding.Another Gene Replacement can be deleted or used to the part of the genomic DNA of the specific polypeptide of coding, but monitoring is that integrate, the gene coding selection marker such as can be used for.Usually, comprise the unaltered flanking DNA of thousands of bases (5 ' and 3 ' end have) (about the description of homologous recombination vector referring to for example Thomas andCapecchi, Cell, 51:503 (1987)) in carrier.Carrier is imported to embryonic stem cell line (for example passing through electroporation), and select wherein the cell (referring to for example Li et al., Cell, 69:915 (1992)) of the DNA that imports and interior source DNA generation homologous recombination.Then for example selected cell is expelled to, in the blastocyst of animal (mice or rat) to form aggregation chimera (referring to for example Bradley, in " Teratocarcinomas andEmbryonic Stem Cells:A Practical Approach ", E.J.Robertson compiles, IRL, Oxford, 1987, the 113-152 pages).Then chimeric embryo can be implanted to the female replace-conceive animal of suitable pseudo-fetus, and make the mature generation of embryo " knock out " animal.The offspring who comprises homologous recombination DNA in its sexual cell can identify by standard technique, and all comprises the animal of homologous recombination DNA for breeding all cells of animal wherein.Knock-out animal can for example have the ability of resisting some pathological condition and form sign aspect pathological condition owing to lacking this polypeptide.

In another embodiment of the invention, provide the method for using antibody in the diagnostic assay method.For example, can in the diagnostic assay method, with antibody, detect expression or the excessively expression of DR5 in specific cells and tissue.Can use multiple diagnostic assay technology known in the art, such as in-vivo imaging algoscopy, external competitive binding assay method, direct or indirect sandwich algoscopy and immunoprecipitation assay, carry out (Zola with out-phase or homophase (heterogeneous or homogeneous phase), Monoclonal Antibodies:A Manual of Techniques, CRC Press, Inc. (1987) pp.147-158).But the antibody used in the diagnostic assay method can be used detection module (moiety) labelling.But detection module should directly or indirectly produce detectable signal.For example, but detection module can be radiosiotope, such as ³h, ¹⁴c, ³²p, ³⁵s or ¹²⁵i; Fluorescence or chemiluminescence compound, such as Fluorescein isothiocyanate, rhodamine or fluorescein; Or enzyme, such as alkali phosphatase, beta galactosidase or horseradish peroxidase.But can adopt that this area knows for any method with the detection module coupling by antibody, comprise those methods of putting down in writing in following document: Hunter et al., Nature 144:945 (1962); David et al., Biochemistry13:1014-1021 (1974); Pain et al., J.Immunol.Meth.40:219-230 (1981); Nygren, J.Histochem.and Cvtochem.30:407-412 (1982).

DR5 antibody also can be used for from recombinant cell culture thing or natural origin affinity purification DR5.In this process, use the method well-known in the art will be for the antibody immobilization of DR5 on suitable holder, such as Sephadex resin or filter paper.The sample that then makes the immobilized antibody contact contain DR5 to be purified, then use suitable solvent clean holder, and described solvent will be removed all substances except DR5 in sample basically, and DR5 is attached on immobilized antibody.Finally, with another kind of suitable solvent clean holder, described solvent will discharge DR5 from antibody.

In another embodiment of the invention, goods and test kit are provided, wherein comprise the material that can be used for treating pathology illness or detection or purification DR5 antibody.Goods comprise the container with label.Suitable container comprises for example medicine bottle, pencil and test tube.Container can be made with various materials, such as glass or plastics.Compositions containing activating agent is housed in container, and described activating agent is effectively treated pathology illness or detection or purification DR5.Activating agent in compositions is DR5 antibody, preferably includes the monoclonal antibody special to DR5.Label on container indication said composition is used for the treatment of pathology illness or detection or purification DR5, but also in can indication body or the usage of external use, all as described above those.

Test kit of the present invention comprises container mentioned above and the second container of buffer agent is housed.It can also comprise other material required on business and user's position, comprises other buffer agent, diluent, filter, syringe needle, syringe and is printed on the package insert of operation instructions.

Provide hereinafter embodiment just to the illustration purpose, but not intention limit the scope of the invention by any way.

All referenced patent of quoting in this complete this description of income and document are as a reference.

Embodiment

Unless otherwise indicated, the commercialization reagent of mentioning in embodiment is used according to the description of manufacturer.The source of those cells of hereinafter differentiating with the ATCC numbering in embodiment and entire description is American type culture collection (American Type Culture Collection, Manassas, Virginia).Much reagent disclosed herein and scheme have further discussion in WO99/37684, WO00/73349, WO98/32856, WO98/51793 and WO99/64461, in this its content of complete income as a reference.

Embodiment 1: the design of Anti-DR5 antibody variant and test

Anti-DR5 antibody 16E2 is derived from show the scFv in storehouse from people's antibody phage, and has been recorded in disclosed WO98/51793 on November 19th, 1998 (seeing embodiment 14).The nucleotide of scFv16E2 and aminoacid sequence are shown in Fig. 5 (SEQ ID NO:9) and Fig. 6 (SEQ ED NO:10).In Fig. 6, signal sequence and heavy chain and light chain CDR district (CDR1, CDR2 and CDR3 district indicate underscore) have been identified.

materials and methods

the structure of total length Anti-DR5 antibody 16E2

Hereinafter described embodiment needs total length IgG.Therefore, the variable region of 16E2 is cloned in the pRK carrier of previous record, this carrier is suitable for the mammalian cell expression (Gormanet al., DNA Prot.Eng.Tech.2:3-10 (1990)) of total length IgG1 antibody.The aminoacid sequence of 16E2 variable region and Kabat data base (Kabat et al., Sequences of Proteins of Immunological Interest, U.S.Dept.of Health and Human Services, NIH, relatively pointing out 5th edition), the variable region of light chain of 16E2 (VL) derived from human lambda light chain family.Therefore, at first the variable region of 16E2 is subcloned in the carrier that contains the λ constant region.Design PCR primer is to add restriction enzyme sites SfiI and MscI, then with these two kinds of variable regions that the enzymic digestion amplification obtains.This fragment is inserted to the carrier that comprises the λ constant region through similar digestion.Because this carrier is in order to design at expression in escherichia coli Fab, for IgG, express, the complete light chain of pcr amplification coding region again, the primer in 5 of coding region ' end adds restriction site AgeI, at 3 ' end interpolation restriction site HindIII.Then, this AgeI-HindIII fragment is inserted to the carrier pDR1 (Clontech) through similar digestion.The complete sequence of plasmid pDR1 is shown in Figure 11 (SEQ ID NO:15).

For the heavy chain of pattern 1, the variable region of heavy chain of pcr amplification scFv16E2 (VH), the primer is designed to add the PvuII site at 5 of described domain ' end, at 3 ' end, adds the ApaI site.Then this fragment is cloned into to the PvuII/ApaI site of carrier pDR2 (Clontech), for the expressed heavy chain (VH-CH1-CH2-CH3 domain).The complete sequence of plasmid pDR2 is shown in Figure 12 (SEQ IDNO:16).

The nucleotide of full length antibody 16E2 heavy chain and light chain and aminoacid sequence are shown in Fig. 7-10 (SEQ ID NO:11-14).Particularly, Fig. 7 and 8 (SEQ ID NO:11 and 12) has shown aminoacid and the nucleotide sequence of total length 16E2 heavy chain, and Fig. 9 and 10 (SEQ ID NO:13 and 14) has shown aminoacid and the nucleotide sequence of total length 16E2 light chain.Heavy chain and the light chain of total length 16E2 antibody hereinafter will be referred to as " pattern 1 ".

the structure of IgG variant

Use direct mutagenesis (Kunkel et al., Proc.Natl.Acad.Sci.USA82:488-492 (1985)) respectively light chain or heavy chain to be built to variant.The pDR2 of the plasmid pDR1 of encoded light chain pattern 1 or encoding heavy chain pattern 1 is transformed into to coli strain CJ236 (BioRad, Joyce andGrindley, J.Bacteriol.158:636-643 (1984)) in, for the preparation of the single stranded DNA template containing BrdU.The aliquot of mutagenesis reaction is transformed in coli strain XL-1 Blue (Stratagene, San Diego, CA), for the purification double-stranded DNA.For every kind of variant, use ABI377x1 or ABI3730x1 automatization DNA sequencer (Perkin-Elmer Corp.), to the complete order-checking of DNA of coding light chain or heavy chain.

For every kind of IgG variant, arrive by the plasmid that will express light chain and the plasmid co-transfection of expressing heavy chain human embryonic kidney cell line 293 (the Graham et al. transformed through adenovirus, J.Gen.Virol.36:59-74, (1977)) in, carry out transient transfection.In brief, by 293 cells sub-bottle that day (split) before transfection, and coating (plate) in containing blood serum medium.Next day, together with pAdVantage ^tMdNA (Promega, Madison, WI) together, prepares the calcium phosphate precipitation thing from the double-stranded DNA of light chain and heavy chain, and dropwise is added in plate.Cell is incubated overnight in 37 ℃, then with PBS, cleans and cultivate 4 days in serum-free medium, now gather in the crops conditioned medium.Use protein A-Sepharose CL-4B from the culture supernatants antibody purification, buffer-exchanged is to the 10mM sodium succinate, and 140mM NaCl, in pH6.0, and used Centricon-10 (Amicon) concentrated.Absorbance by measuring the 280nm place or measure protein concentration by quantitative amino acid analysis.

electrochemiluminescence DR5 binding assay

Measure the relative combination of Anti-DR5 antibody with liquid phase competitive ELISA form.The DR5-Fc fusion rotein is used biotin-X-NHS (Research Organics, Cleveland, OH) biotinylation, ORI-TAG NHS ester (IGEN Intemational for standard antibody (or pattern 1 or Apomab 7.3), Gaithersburg, MD) according to the description labelling of manufacturer.In order to carry out binding assay, by the serial dilution in measuring buffer (PBS, pH7.4, containing 0.5%BSA and 0.5%Tween-20) of test antibody sample.Antibody sample (concentration range 50 by equal-volume (each 25 μ l), 000-0.85ng/ml), ORI-TAG standard antibody (150ng/ml) and biotinylated people DR5-Fc (15ng/ml) be added in 96 hole polypropylene boards, in room temperature, shaking gently lower incubation 1.5 hours.Then add streptavidin magnetic bead (IGEN International) (25 μ l/ hole), by as above incubation 30 minutes again of plate.Add and measure buffer to every hole final volume 250 μ l, use ORIGEN M384 instrument (IGEN International) to the plate reading.Use four parameter fittings (four parameter fit) of sample curve to calculate the IC50 value.

bioassary method: growth of tumour cell suppresses/kills and wounds

Measure in vitro the apparent effect of every kind of antibody variants in the tumor cytotoxicity algoscopy.Cultivate the Colo205 CCL188 in containing the RPMI culture medium of 10% hyclone.In being housed, 96 hole tissue culturing plates of culture medium carry out 2 times of serial dilutions of standard substance (or pattern 1 or Apomab 7.3) and sample, contain or do not contain cross-linking antibody (anti-human Fc, the F of goat affinity purification (ab ') of 10 μ g/ml concentration in described culture medium ₂).Then cell (20,000/ hole) is added in plate.By plate in 37 ℃ of incubations 48 hours altogether.Within last 3 hours of incubation, in hole, add Alamar Blue.Use exometer to the fluorescence reading, excite as 530nm, be emitted as 590nm.Use four parameter curve program analysis data.

result

The overall goal of this work is that exploitation has the effect of the biochemical characteristic of improvement and improvement and do not damage the Anti-DR5 antibody of safety.

With several methods realize the improvement of expectation comprising that amino acid replacement in DR5 heavy chain and light chain is to improve chemical stability or heat stability; Folding; The Alanine-scanning of CDR residue with determine which residue in conjunction with or folding may be important, therefore can change in order to improve affinity; And identify the clone with improved affinity with phage display CDR storehouse.Also on the Changeement in framework region they may affect immunogenicity and biologic activity.

Use homology model (Figure 19 and 20) to help select many these changes.

the heavy chain variant

Series 1

5 places that the heavy chain of pattern 2 comprises with respect to pattern 1 change.These changes (Q6E, V11L, E12V, R13Q and K105Q) are arranged in the framework of variable region, are in order to make framework more approach people V _hthe III consensus sequence adds.Table 1 has shown the variant at first built, and they change in heavy chain CDR.The ssDNA template of these mutants is patterns 2.It is in order to improve packing (packing) that the 102nd Leu becomes Tyr, and improves thus stability.Change and combine therewith, Asn53 becomes Gln or Tyr is in order to eliminate potential deacylated tRNA amine site.It is in order to eliminate potential oxidation site that Met34 becomes Leu.These heavy chain variants are expressed with together with initial light chain, produce pattern 20-23 (table 1).After this heavy chain that comprises the framework change in three sudden change M34L, the N53Q of place and L102Y and pattern 2 is called " tripleheavy one " or TH1, and is called TH2 like the heavy chain class of the framework that comprises three sudden change M34L, the N53Y of places and L102Y and pattern 2.

Table 1: heavy chain CDR variant

Pattern a	Substitute	IC50 variant IC50v1 b	Crosslinked bioassay activity is arranged c	Without crosslinked bioassay activity d
					20	N53Q，L102Y	0.11	0.24	*
21	M34L，N53Q，L102Y	0.42	0.54	＜1
					22	N53Y，L102Y	0.08	1.30	Faint
23	M34L，N53Y，L102Y	0.28	4.47	Nothing

A template: pattern 2

BOrigen

competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

Because combination after interpolation M34L sudden change has loss with the cell in vitro killing activity, (be that v21 compares with v20, v23 compares with v22, table 1), use heavy chain pattern 20 as template, carried out a series of other sudden changes in heavy chain CDR1, substituted with alanine or other residue of drawing by scanning Kabat data base.Together with the light chain of these heavy chains and pattern 1, express.Table 2 shown the sudden change of these patterns aminoacid, obtain with respect to v1 in conjunction with, and bioassary method data.Gly33 becomes Ala and improves in conjunction with and improve effect.This heavy chain that comprises three sudden change G33A, the N53Q of place and L102Y is called TH3.Equally, built the TH4 that comprises G33A, N53Y and L102Y.Thr28 becomes Ala and compares also raising activity with v1, and the heavy chain that comprises T28A, N53Q and L102Y is called TH9.The CDR that table 5 has been summed up in TH1, TH2, TH3, TH4 and TH9 changes.These 5 kinds of heavy chains make further research after with light chain, combining (seeing below) coexpression.

Variant in table 2:CDR H1

Pattern a	Sudden change	IC50 mutant IC50v1 b	Crosslinked bioassay activity is arranged c	Without crosslinked bioassay activity d
					111	G26A	0.11	0.42	Nothing
	F27A	ND	ND	ND
					112	T28A	0.19	0.8	Identical with v1
127	F29A	7.6	Nothing
					55	D30A	2.2	ND	ND
54	D308	2.1	ND	ND
					57	D31A	＞10	ND	ND
56	D31S	4.3	ND	ND
					113	Y32A	1.65	＞100	Nothing
58	G33A	0.1	0.097	Faint
					59	M34A	0.8	3.49	Faint
49	M34I	1.2	14.6	Nothing
					50	M34S	1.0	13.8	Nothing
	S35H	ND	ND	ND
					130	S35A	ND	ND	ND

The template of all variants of a is all pattern 20

BOrigen competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

Alanine-scanning CDR H2 and CDR H3

In order further to illustrate each amino acid whose contribution in heavy chain CDR2 and CDR3, by each substituting in these residues with alanine, built mutant.Use heavy chain pattern 1 as template, with the alternative synthetic oligonucleotide of coding alanine, carry out direct mutagenesis.These heavy chains are expressed together with pattern 1 light chain.The apparent affinity of gained antibody and the variation of effect have been shown in table 3 and 4.Gly99Ala and Arg100Ala improve activity, so these changes can be used for building the other combination mutant of heavy chain.

The Alanine-scanning of table 3:CDR H2

Pattern a	Sudden change	Combining ratio with respect to v1 b	Crosslinked bioassay activity is arranged c	Without crosslinked bioassay activity d
					139	G50A	3.55	1.91	Nothing
140	I51A	ND	ND	ND
					141	N52A	2.42	ND	ND
	W52aA	ND	ND	ND
					142	N53A	ND	0.19	Faint
160	G54A	1.49	ND	ND
					151	G55A	1.0	0.86	Low over 100 times
143	S56A	1.1	0.81	Approximate v1
					144	T57A	1.2	0.54	Low over 100 times
152	G58A	0.55	1.24	Low approximately 5 times
					153	Y59A	2.22	3.42	Nothing
154	A60A	0.55	0.49	Low over 100 times
					158	D61A	0.725	ND	ND
155	S62A	0.65	0.73	Low over 100 times
					156	V63A	0.041	ND	Low approximately 3 times
159	K64A	ND	ND	ND
						G65A	ND	ND	ND

A template: pattern 1

BOrigen

competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

Alanine mutation body in table 4:CDR H3

Pattern a	Sudden change	Combining ratio with respect to v1 b	Crosslinked bioassay activity is arranged c	Without crosslinked bioassay activity d
					108	K102A	1.82	＞10	Nothing
109	I95A	8.38	Nothing	Nothing
					(1040)	L96A	ND	ND	ND
126	G97A	8.52	Nothing	Nothing
					110	Y98A	24.2	Nothing	Nothing
128	G99A	0.09	0.43	High approximately 8 times
					129	R100A	0.47	0.128	High approximately 4 times
116	G100aA	2.84	Nothing	Nothing
					117	W100bA	Na	Nothing	Nothing
118	Y100cA	10.85	Nothing	Nothing
					119	F100dA	Na	Nothing	Nothing
120	D101A	1.83	～1	Nothing

A template: pattern 1

BOrigen

competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

Series 2

Built the 2nd serial heavy chain that uses identical CDR with pattern TH1, TH2, TH3 and TH4, its middle frame residue E6, L11, V12, Q13 and Q105 revert back to the aminoacid of finding in pattern 1, i.e. Q6, V11, E12, R13 and K105.These heavy chains are called TH5, TH6, TH7 and TH8 (in Table 5).

Table 5: the heavy chain variant that sudden change is arranged in all CDR rings

The heavy chain combination	Sudden change in CDR H1	Sudden change in CDR H2	Sudden change in CDR H3	6, the framework residue at 11,12,13,105 places
					TH1	M34L	N53Q	L102Y	E，L，V，Q，Q
TH2	M34L	N53Y	L102Y	E，L，V，Q，Q
					TH3	G33A	N53Q	L102Y	E，L，V，Q，Q
TH4	G33A	N53Y	L102Y	E，L，V，Q，Q
					TH5	M34L	N53Q	L102Y	Q，V，E，R，K
TH6	M34L	N53Y	L102Y	Q，V，E，R，K
					TH7	G33A	N53Q	L102Y	Q，V，E，R，K
TH8	G33A	N53Y	L102Y	Q，V，E，R，K
					TH9	T28A	N53Q	L102Y	E，L，V，Q，Q

the light chain variant

The Alanine-scanning of light chain CDR

In order better to understand the contribution of light chain CDR residue to combination and biologic activity, use direct mutagenesis that each aminoacid is become to alanine.By each light chain variant and heavy chain pattern 1 (V1) combination, for the transient expression of IgG as above.Table 6 has been summed up the result of light chain CDR Alanine-scanning.What is interesting is, as if contrary with much other antibody, CDR L1 plays a significant role in the antigen combination.This light chain is the λ chain, and the CDR1 of specification of a model shown in Figure 20 can form the α spiral.More can tolerate that the residue in L2 and L3 substitutes with alanine, except the G50A in CDR2, it eliminates combination.On the contrary, some alanine substitutes, and especially R91A and K51A, improve combination and biological activity.

Table 6: light chain Alanine-scanning mutant

Pattern a

Position

Sudden change

In conjunction with b

Bioassay c

Bioassay d

89

CDRL1

Q24A

1.22

0.87

*

40

CDRL1

G25A

2.54

ND

ND

90

CDRL1

D26A

0.76

0.88

Nothing

41

CDRL1

S27A

2.36

2.79

Faint

42

CDRL1

L28A

＞100

ND

ND

46

CDRL1

R29A

3.0

ND

ND

38

CDRL1

S30A

2.35

5.51

N/A

39

CDRL1

Y31A

＞10

ND

ND

47

CDRL1

A33G

6.4

ND

ND

43

CDRL1

S34A

1.54

3.23

Faint

64

CDRL2

G50A

＞1000

ND

ND

65

CDRL2

K51A

0.4

0.027

***

93

CDRL2

N52A

3.12

ND

ND

94

CDRL2

N53A

7.54

ND

ND

95

CDRL2

R54A

0.89

0.87

*

107

CDRL2

P55A

0.95

1.35

Faint

163

CDRL2

S56A

1.9

ND

ND

72

CDRL3

N89A

3.1

Na

Nothing

73

CDRL3

S90A

0.9

0.91

1.0

74

CDRL3

R91A

0.5

0.098

****

164

CDRL3

D92A

0.22

ND

ND

137

CDRL3

S93A

2.51

0.85

Faint

138

CDRL3

S94A

0.31

0.15

Faint

165

CDRL3

G95A

0.06

ND

ND

71

CDRL3

N95aA

1.0

1.17

ND

(1024)

CDRL3

H95bA

ND

ND

ND

166

CDRL3

V96A

1.34

ND

ND

167

CDRL3

V97A

1.05

ND

ND

A template: pattern 1

BOrigen

competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

Gene family residue exchange (Gene family residue swap)

Study light chain with the orthomutation body of the second type.In the method, by showing as the either side that is arranged in ring in model, therefore may bring into play supporting function but not the CDR residue that is directly involved in the antigen combination is exchanged into the residue of other closely related λ gene family correspondence position.Also these mutants are expressed together with the v1 heavy chain, summed up result in table 7.In CDR L2, G50K, K51D be combined in combination and two aspects of biological activity all produce remarkable improvement, the combination that comprises the G50K that suddenlys change, K51D, N52S, N53E everywhere also improves to some extent with respect to v1.Can not tolerate in (tolerate) the same area and only involve other more conservative change that place's residue substitutes.

Table 7: the light chain variant based on related gene man family sequence

Pattern a	Position	Sudden change	In conjunction with b	Bioassay c	Bioassay d
						25	CDRL1	Q24S，D26E，Y31K，S34Y	11.7	ND	ND
24		D2GE，Y31K	＞100	ND	ND
						51		S34Y	＞100	ND	ND
44		Q24S	0.96	1.23	Faint
						45		Y31K	＞1000	ND	ND
106		Y32H	5.1	1.71	Nothing
						26	CDRL2	G50K，K51D，N52S，N53E	0.33	0.071	1
27		G50S，KS1D，N52S	0.92	0.29	Faint
						91		G50K，K52S，N53E	14.71	ND	ND
92		G50K，K51D	0.09	0.43	***
						28		N52L	6.88	ND	ND
125		G50S，KS1D，N52S，N53E	1.99	1.0	ND
						32		N52Q	5.28	ND	ND
33		NS3Q	4.48	ND	ND
						61		N52S，N53E	＞100	ND	ND
62		N52S	1.1	1.1	Nothing
						63		N52Q，N53S	＞10	ND	ND
60	CDRL3	N89L，R91A，N95aT，H95bY	＞100	ND	ND
						52		N95aT，H95bY	2.3	ND	ND
30		N95aQ	1.65	7.3	Faint
						31		N89Q	＞1000	ND	ND
29		H95bY	1.68	2.14	Faint
						66		H95bR	0.7	0.97	1.0
67		N95aK	1.0	0.44	1.0

A template: pattern 1

BOrigen

competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

The affinity carried out with phage antibody library is selected

To each CDR, L1, L2 and L3, build respectively phage display library and select the clone to the affinity raising of DR5-Ig.The inspection of model (Figure 20) is pointed out to which residue in CDR likely exposes, and selects these residues to carry out randomization.The complete lambda light chain of pattern 1 and VH domain are cloned in Vector for Phage Display pS1602, referring to Vajdos et a1., J.Mol.Biol.320:415-428 (2002), be further described in Sidhu et al., Curr.Opin.Biotechnol.11:610-616 (2002).Use Kunkel mutation in library construction.The v1 phasmid is transformed in coli strain CJ236, for the preparation of single stranded DNA, and uses the oligonucleotide that comprises the TAA codon in each site of selecting for randomization to produce the library template.Then use and utilize the oligomer of degenerate codon NNS (wherein N is the geometric ratio mixture of G, A, T and C, and S is the geometric ratio mixture of G and C) to build library.CDRL1 suddenlys change with stopping template oligomer CA945 and library oligomer CA946.CDRL2 suddenlys change with stopping template oligomer CA947 and library oligomer CA948.CDRL3 suddenlys change with stopping template oligomer CA949 and library oligomer CA950.Summed up library construction in table 8.

Table 8

The oligomer numbering	District	Purpose	Sequence
				CA945	CDRL1	Stop template	CAT GCC AAG GAG ACT AAC TCA GAT AAT ATT AAG CTA GCT GGT ACC AGC (SEQ ID NO：21)
CA946	CDRL1	Randomization	CAT GCC AAG GAG ACN NSC TCA GAN NST ATN NSG CTA GCT GGT ACC AGC (SEQ ID NO：22)
				CA947	CDRL2	Stop template	GTC ATC TAT GGT AAA TAA TAA CGG CCG TCT GGC ATC CCA GAC CG(SEQ ID NO：23)
CA948	CDRL2	Randomization	CTT GTC ATC TAT GGT AAA NNS NNS NNS CCG TCT GGC ATC CCA GAC CG (SEQ ID NO：24)
				CA949	CDRL3	Stop template	GCT GAC TAT TAC TGT AAC TCC CGG TAA TAA TAA GGC TAA CAT GTG GTA TTC GGC GGA GG(SEQ ID NO：25)
CA950	CDRL3	Randomization	GCT GAC TAT TAC TGT AAC TCC CGG NNS NNS NNS GGC NNS CAT GTG GTA TTC GGC GGA GG(SEQ ID NO：26)

The product electroporation of random mutagenesis reaction, in XL1-Blue Bacillus coli cells (Stratagene), is increased by cultivate 14-16 hour together with the M13K07 helper phage.Assess storage capacity (library size) by serial dilution and coated plate, suppose to be applied on the Carbenicillin plate by initial conversion product and be 1.9 * 10 ⁹to 2.2 * 10 ⁹individual clone.

With liquid phase, use biotinylated DR5-Ig (Genentech) elutriation 4 to take turns in library.By about 10 ¹¹3% defatted milk powder in 1ml PBS for individual phage, 0.2%Tween solution (phage confining liquid) seals 1 hour in room temperature on swiveling wheel.DR4-IG and CD4-IG are added in confining liquid to reduce non-specific binding with 1 micromole separately.Then the first round adds biotinylated antigen with 100nM, allows in conjunction with carrying out 2 hours.In the elutriation of subsequent passes, antigen concentration is down to 10,5 and 1 nanomole.

In order to catch the phage of conjugated antigen, at first the magnetic bead (Dynal) that is coated with streptavidin is cleaned 3 times with the phage confining liquid, then with 1ml phage confining liquid, in room temperature, seal 1 hour.Magnetic bead is concentrated and is added in antigen-phage solution with magnet and reach 15 minutes.Then with magnetic bead, magnetic bead-antigen-phage complex is pulled out to solution.Then these magnetic beads are cleaned 3 times with the phage confining liquid, clean 3 times with PBS-Tween (0.02%Tween), and clean 1 time with PBS.By with 100 μ l 0.1MHCl, soaking 10 minutes wash-out bacteriophages from magnetic bead, and neutralize with NaOH.By the phage of eluting, for infecting XL1 Blue escherichia coli, breeding is for subsequent passes as mentioned above.Each is taken turns and improves the stringency of cleaning.

To the cloning and sequencing from each library.For the L1 library, have to wild-type sequence, support that this CDR is important idea for antigen combination or antibody conformation, tolerable only a few sudden change in potential spiral.For the library in L2, do not find consensus sequence, illustrate that multiple CDRL2 sequence is for being acceptable in conjunction with DR5.For the L3 library, several sequences occur repeatedly, use the mutation of oligomer guidance that these sequences are transplanted on the full-length light chains carrier.Again, at 293 cells IgG, use heavy chain pattern 1 to carry out cotransfection.Table 9 has been described the L3 sequence that is expressed as full length antibody and the result of combination and bioassary method.Mix pattern 69 and produce with two kinds of row that check order of 70 antibody of comparing combination and biological activity raising with pattern 1.

Table 9: derived from the protein sequence of the light chain CDR3 of phage library

Pattern a	CDR L3 sequence	Combining ratio with respect to v1 b	Crosslinked bioassay activity is arranged c	Without crosslinked bioassay activity d
					68	NSRDSSGSHVV	1.3	0.973	1.0
69	NSRSYSGNHVV	0.1	0.143	****
					70	NSRSSSGSHVV	0.2	0.152	***

A template: pattern 1

BOrigen

competitive people DR5 binding assay

C inhibiting tumour cells algoscopy

D inhibiting tumour cells algoscopy

The combination light chain

As mentioned above, identified the sudden change that improves alone external combination and tumor cytotoxicity in each light chain CDR.Then combining these suddenlys change to produce in several each CDR improved light chain is arranged.These " triple light1 " etc. are called TL1, TL2 and TL3, are described in table 10.So, the combination that the L3 identified in the L2 sudden change of identifying in the L1 sudden change that TL1 comprises evaluation in pattern 44, pattern 26 and pattern 29 suddenlys change.Equally, TL2 comprises from the L1 of pattern 44 sudden change, from the L2 sudden change of v65 with from the combination of the L3 sudden change of v69, and TL3 comprises L1 from v44, from the L2 of v65 with from the combination of the L3 of v74.

Table 10: the sudden change in the light chain combination

The light chain combination	Sudden change, CDRL1	Sudden change, CDRL2	Sudden change, CDRL3
				TL1	Q24S	G50K，K51D，N52S，N53E	H95bY
TL2	Q24S	K51A	D92S，S93Y
				TL3	Q24S	K51A	R91A

Table 11:Apomab nomenclature

Light chain:	TL1	TL2	TL3
				Heavy chain
TH1	Apomab 1.1	Apomab 1.2	Apomab 1.3
				TH2	Apomab 2.1	Apomab 2.2	Apomab 2.3
TH3	Apomab 3.1	Apomab 3.2	Apomab 3.3
				TH4	Apomab 4.1	Apomab 4.2	Apomab 4.3
TH5	Apomab 5.1	Apomab 5.2	Apomab 5.3
				TH6	Apomab 6.1	Apomab 6.2	Apomab 6.3
TH7	Apomab 7.1	Apomab 7.2	Apomab 7.3
				TH8	Apomab 8.1	Apomab 8.2	Apomab 8.3
TH9	Apomab 9.1	Apomab 9.2	Apomab 9.3

Shown other Apomab antibody in table 12

The Apomab pattern	6th, the framework amino acid sequence at 11,12,13 and 102 places	To the combination of DR5, with respect to Apomab 7.3	Effect, with respect to Apomab 7.3, have crosslinked	The bioassay activity, have crosslinked
					7.3 *	Q VER K	1.00	1	ND
3.3 **	E LVQ Q	2.50	0.53	Nothing
					18.3	E LVQ K	0.51	0.75	Nothing
24.3	E VER Q	0.80	1	Nothing
					25.3	Q LVQ K	0.77	1	Low approximately 3 times
26.3	Q VER Q	0.53	1	Low approximately 6 times
					27.3	E VER K	1.90	1.2	Nothing
28.3	Q LER K	0.48	0.94	Low approximately 5 times
					29.3	Q LEQ K	0.52	1.2	Low approximately 7 times
30.3	Q WQ K	0.78	0.66	High approximately 3 times
					36.3	Q VVR K	0.66	ND	ND
37.3	Q VEN K	0.84	ND	ND
					38.3	Q LVR K	0.47	ND	ND
18.2	E LVQ K	0.11	0.16	Nothing
					24.2	E VER Q	0.14	0.18	Nothing
25.2	Q LVQ K	0.10	0.13	High approximately 32 times
					26.2	Q VER Q	0.14	0.22	High approximately 10 times
27.2	E VER K	0.10	0.15	Nothing

^*framework is identical with pattern 1

^*framework is identical with the total VH-III of people

Apomab expresses

After deriving triple heavy chain and triple light chain, produced the combinatorial antibody of 9 * 3 grids (grid) by each the cotransfection in each and 9 kinds of heavy chains in 3 kinds of light chains in 293 cells, and purification gained antibody as mentioned above.Use the in vitro study of these Apomab to point out that several versions is quite effective in bioassary method.Therefore, preparation is suitable for the material of mouse tumor model research in body.

Apomab reclaims and purification

Method for the recovery of the cell culture fluid (HCCF) from results and purification Apomab antibody is described below:

The cell culture fluid (HCCF) of the results of Prosep protein A chromatography-will be produced by Chinese hamster ovary (CHO) cell is adjusted to 7.0 with 1.5M Tris alkali by pH, be loaded into the EDTA with 25mMNaCl/25mM Tris/5mM, on the Prosep protein A post (Millipore, USA) of pH7.5 balance.By cleaning with level pad, then with the 0.5M TMAC in level pad, clean second time, more then clean the 3rd time with level pad, make uncombined protein flow through and remove.Use the ladder eluting (step elution) of 0.1M acetic acid that Apomab antibody is eluted from the protein A post.Monitor the post eluent by A280.Merge Apomab antibody peak.

SP-Sepharose Fast Flow (speed stream) chromatography-will with 1.5M Tris alkali, pH be adjusted to 5.5 from the amalgamation liquid of the Apomab antibody of Prosep protein A, then be loaded on the SP-Sepharose Fast Flow post (Amersham Pharmacia, Sweden) by 25mM MOPS pH7.1 balance.After load sample, post is cleaned to A280 and reaches baseline with level pad.0-0.2M NaCl linear gradient by the level pad pH8 that uses 12 times of column volumes, elute Apomab antibody from post.Monitor the post eluent by A280.Collect fraction and merge those fraction that the mensuration of analyzing according to SEC-HPLC contains correct folding Apomab antibody.

Q-Sephrose Fast Flow chromatography-then SP-Sepharose fraction amalgamation liquid is loaded on the Q-Sepharose FF post (Amershampharmacia, Sweden) by 50mM NaCl/25mM Tris pH of buffer 8 balances.Clean pillar with level pad, collect the Apomab antibody in the post eluent.

UF/DF preparation-in the molecular weight cutoff value, approximately on 10,000 daltonian films, by ultrafiltration, concentrate the Q-Sepharose amalgamation liquid.Then by the Q Sepharose FF amalgamation liquid after concentrating to the 10mM histidine of 10 times of volumes/8% sucrose, pH6 diafiltration.Regulate Q Sepharose amalgamation liquid after (condition) diafiltration to the final concentration of polysorbate20 with 10% polysorbate20 and reach 0.02%.The liquid in bulk (bulk) prepared is filtered through aseptic 0.22 μ m filter, be stored in 2-8 ℃ or-70 ℃.Measure the final purity of Apomab antibody by SDS-PAGE, SEC-HPLC and amino acid sequence analysis.

Order-checking is carried out on ABI3700 or ABI3730 Applied Biosystems sequenator.The order-checking tomographic map is used Sequencer (GeneCodes, Ann Arbor, MT) sequence analysis software to be analyzed.

The external activity of test Apomab

Before research, each batch tested to external activity as mentioned above in the body that starts Apomab.These the results are shown in table 13.

The external activity of table 13:Apomab

The Apomab pattern	The multiple that apparent affinity improves with respect to pattern 1 (v1)	Have crosslinked, the multiple that apparent effect improves with respect to pattern 1 (v1)	Without crosslinked, with respect to the apparent effect of pattern 1 (v1)
				1.1	11.30	24	Nothing
1.2	52.10	14	Nothing
				1.3	13.10	6.5	Nothing
2.1	12.80	5.5	Nothing
				2.2	9.60	3	Nothing
2.3	29.00	6.5	Nothing
				3.1	15.00	14	Nothing
3.2	29.20	22	Nothing
				3.3	15.00	8	Nothing
4.1	25.70	11	Nothing
				4.2	23.10	6	Nothing
4.3	10.30	12	Nothing
				5.2	26.90	22	High approximately 8 times
5.3	2.80	10	Low approximately 2.4 times
				6.2	31.60	6	High approximately 2 times
6.3	1.50	4	Low approximately 4 times
				7.2	31.20	22	High approximately 5 times
7.3	4.30	16	Low approximately 2 times
				8.2	28.60	9	Very faint
8.3	8.30	11	Low approximately 8 times
				9.1	21.90	36	Nothing
9.2	29.90	48	Nothing
				9.3	10.00	ND	Nothing

Embodiment 2: the anti-tumor activity of assessment Apomab in other xenograft models of Colo205 human colon carcinoma xenograft models and colorectal carcinoma

The abbreviation commonly used of using in this embodiment and subsequent embodiment is as follows:

CR disappear fully (complete regression)

PR partly disappear (partial regression)

MTD maximum tolerated dose (maximum tolerated dose)

MTV intermediate value gross tumor volume (median tumor volume)

The non-treatment associated death of NTR (non-treatment related death)

LTTFS is for a long time without tumor survival person (long-term tumor-free survivor)

PBS phosphate buffered saline (PBS) (phosphate-buffered saline)

Q3dx4 every 3 days 1 time, totally 4 doses (once every three days for a total of four doses)

Qdx1 gives 1 dose (one dose given on Day1) on the 1st day

Qdx5 every day 1 time, continue 5 days (once daily for five days)

TFS is without tumor survival person (tumor-free survivor)

TR treats associated death (treatment related death)

The TTE time (time to endpoint) to terminal

The difference of intermediate value TTE value between the animal that T-C receives treatment and control animal, in sky

Tumor growth delay in TGD (tumor growth delay); T-C; The group of receiving treatment and right

Compare the increase of intermediate value TTE according to group and usually state the % contrast as

The Time to 2xVo doubling time (doubling time, (DT)); Gross tumor volume doubles the spent time

LogCellKill=log ₁₀(V _before/ V _after), actual gross tumor volume log during treatment ₁₀(V _before) and log ₁₀(V _after) between difference (Chenevert et al., Clin.Cancer Res.3:1457-1466 (1997))

Give every mice of 6-8 week female nude mouse in age (Charles River Laboratories) in right drosal part subcutaneous vaccination 0.2ml volume 5 * 10 ⁶individual Colo205 cell.Play ear label (ear-tag) for discriminating to all mices.Once gross tumor volume reaches about 100-200mm ³, will take tumor Colo205 mice random packet and implement treatment.

Therapeutic scheme is intraperitoneal single dose and several doses of media (vehicle) contrast or standard substance and test antibody, 3mg/kg/ mice or 10mg/kg/ mice.In some situation, will within latter 5 minutes, 24 hours or 48 hours, collect serum and tumor in treatment from 3 or 4 mice euthanasia of medium and 10mg/kg group, for serum drug level and tumor histology's research.In remaining mice, measurement of tumor carried out twice weekly at first 2 weeks, then other 4 weeks, weekly.

What in Colo205 xenotransplantation nude mouse model, obtain the results are shown in Figure 21-25.

Figure 21 has shown that Apomab 5.3,6.3 and 8.3 each all dosage in test are reducing aspect mean tumour volume all highly effectively, and their effect is substantially the same with antibody 16E2 pattern 1.

Test the effect of intraperitoneal single dose Apomab 5.2,6.2,5.3,7.2 and 7.3 in Colo205 xenotransplantation nude mouse model, the results are shown in Figure 22.All Apomab of test height aspect the reduction gross tumor volume is effective, and their effect is substantially the same with antibody 16B2 pattern 1.

Similarly, result shown in Figure 23 has shown that Apomab 5.2,7.3 and 8.3 is effective aspect the reduction gross tumor volume in this model of colorectal carcinoma.In this experiment, Apomab and 16E2 use with the dosage of 1mg/kg and 3mg/kg, but the treatment of other side as mentioned above.Apomab 7.3 and 8.3 effect are remarkable especially, and do not show any reverse at the test period of 20 days.

As shown in figure 24, in the Colo205 xenograft models, the active anticancer of Apomab 7.3 surmounts the activity of Apomab 23.3 and 25.3 far away.

Figure 25 has shown relatively the measurement result derived from the anti-tumor activity of Apomab 7.3 in Colo205 mice xenograft models of stable and instantaneous cell line.In brief, as mentioned above, give every mice of 6-8 week female nude mouse in age in right drosal part subcutaneous vaccination 0.2ml volume 5 * 10 ⁶individual Colo205 cell.Dosage is shown in figure.Data show in Figure 25 is effectively equal in Colo205 mice xenograft models derived from the stable Apomab 7.3 with instantaneous cell line respectively.

Figure 26 shown test Apomab 7.3 (10mg/kg dosage) as monotherapy (monotherapy) or with the experimental result of the active anticancer of conjoint therapy (combination therapy) in the HCT15 of colorectal carcinoma xenograft models of 80mg/kgCPT-11 (irinotecan (irinotecan) is used for the treatment of a kind of known drug of colorectal carcinoma).Shown in result prove, although Apomab 7.3 and CPT-11 are effectively when using separately, combine for two kinds and demonstrate remarkable effect, surmount the two activity of using as monotherapy of Apomab 7.3 and CPT-11.

The representativeness experiment of carrying out in carrying sarcoma LS180 xenograft, nude mice with Apomab 7.3 associating CPT-11 (irinotecan) treatment the results are shown in Figure 27.Again, the discovery conjoint therapy is better than using in Apomab 7.3 or CPT-11 arbitrary as single medicament, and difference is significant (the 2nd little figure) statistically.That is compared is all to Tukey-Kramer P<0.05.

Embodiment 3: the active anticancer of assessment Apomab 7.3 in the BJAB of non_hodgkin lymphoma xenograft models

In the BJAB of non_hodgkin lymphoma xenograft models assessment independent with associating RITUXAN

the active anticancer of the Apomab 7.3 (10mg/kg q1 wk) of (rituximab, Genentech.Inc.) (4mg/kg, q1 wk).Because known lymphoma cell is grown better in the SCID mice, so use 6-8 week SCID mice in age (Charles River Laboratory) in this research.Treat parameter and the results are shown in Figure 28.Apomab 7.3 and RITUXAN demonstrate synergistic activity (synergistic activity) when co-administered.

Embodiment 4: the active anticancer of assessment Apomab 7.3 in the BxPC3 of human pancreas's adenocarcinoma xenograft models

In the BxPC3 xenograft models of the human pancreas's adenocarcinoma in female nude mouse (Charles River Laboratory) investigation independent with associating gemcitabine (gemcitabine) (160mg/kg, intraperitoneal) active anticancer of Apomab 7.3 (10mg/kg, intravenous).Treat parameter and the results are shown in Figure 29.The active anticancer of the Apomab 7.3 that data show is used as monotherapy is far superior to the effect of gemcitabine.The co-administered further raising that causes effect of Apomab 7.3 and gemcitabine.

Embodiment 5: the active anticancer of assessing Apomab 7.3 independent and associating carboplatin and taxol in the H460 of people's pulmonary carcinoma xenograft models

With respect to the combination of medium contrast and carboplatin and taxol, assess the active anticancer of Apomab 7.3 (10mg/kg, 1x wk, intraperitoneal) independent and associating carboplatin and taxol.Give every mice of 60 female nude mouses (Charles River Laboratory) in right drosal part subcutaneous vaccination 0.2ml volume 5 * 10 ⁶individual H460 cell.Play the ear label for discriminating to all mices.Allow that tumor reaches mean tumour volume 100-200mm ³and treatment as shown in figure 30.In brief, mice is divided into to four groups.The 1st group is medium treatment matched group (10mM histidine, 8% sucrose, 0.02%Tween20 pH6).With Apomab 7.3, treat for the 2nd group, 10mg/kg/ mice dosage, intraperitoneal, 1 time weekly, continue 2 weeks.Use carboplatin (carboplatin) (100mg/kg/ mice, intraperitoneal, the 0th day single dose)+taxol (taxol) (6.25mg/kg/ mice, subcutaneous, continuous 5 days once a day, continues 2 weeks) for the 3rd group.Accept Apomab 7.3 (10mg/kg/ mice dosage, intraperitoneal, 1 time weekly for the 4th group, continue 2 weeks)+carboplatin (100mg/kg/ mice, intraperitoneal, the 0th day single dose)+taxol (6.25mg/kg/ mice, subcutaneous, continuous 5 days once a day, continues 2 weeks).The anti-tumor activity of the active anticancer of the Apomab 7.3 used as monotherapy and carboplatin+taxol combination is suitable.Co-administered other treatment mode that is better than of Apomab 7.3+ carboplatin+taxol.

Embodiment 6: the active anticancer of assessing Apomab 7.3 independent and associating carboplatin and taxol in the H2122 of people's pulmonary carcinoma xenograft models

In this research, give every mice of female nude mouse (Charles River Laboratory, every group of 10 mices) in right drosal part subcutaneous vaccination 0.2ml volume 5 * 10 ⁶individual H2122 cell.Play the ear label for discriminating to all mices.Allow that tumor reaches mean tumour volume 100-200mm ³, be divided at random four groups (every group of 6 mices), and treatment as shown in figure 31.The 1st group is medium treatment matched group (10mM histidine, 8% sucrose, 0.02%Tween20 pH6).Use carboplatin (100mg/kg/ mice, intraperitoneal, the 0th day single dose)+taxol (6.25mg/kg/ mice, subcutaneous, continuous 5 days once a day, continues 2 weeks) for the 2nd group.Accept Apomab 7.3 (10mg/kg/ mice dosage, intraperitoneal 1 time weekly, continue 2 weeks) for the 3rd group.Accept Apomab 7.3 (10mg/kg/ mice dosage, intraperitoneal, 1 time weekly for the 4th group, continue 2 weeks)+carboplatin (100mg/kg/ mice, intraperitoneal, the 0th day single dose)+taxol (6.25mg/kg/ mice, subcutaneous, continuous 5 days once a day, continues 2 weeks).As shown in figure 31, carboplatin+taxol combination does not show the remarkable active anticancer with respect to medium treatment matched group.Fully contrary, the Apomab 7.3 used as monotherapy demonstrates significant anti-tumor activity.Demonstrate response (CR) fully and without any reverse during treatment in 70 days with all 6 mices of Apomab 7.3 treatment.Find identical activity in the group with Apomab 7.3+ carboplatin+taxol combined therapy.

In order to measure the maximum effective dose of Apomab 7.3 in this model, give every mice of 6-8 week female nude mouse in age (Charles River Laboratory) in right drosal part subcutaneous vaccination 0.2ml volume 5 * 10 ⁶individual H2122 cell.Play the ear label for discriminating to all mices.Allow that tumor reaches mean tumour volume 100-200mm ³, random packet (every group of 13 mices), and treatment as described below.By any mice euthanasia of getting rid of beyond treatment group.

The A group is medium treatment matched group (0.5M succinic acid arginine, 20mM Tris, 0.02%Tween20, pH7.2).The medium intraperitoneal is used, and 5 times weekly, continues 1 week.The B group is used Apomab 7.3,1mg/kg/ mice, intravenous, single dose.The C group is used Apomab 7.3,3mg/kg/ mice, intravenous, single dose.The D group is used Apomab 7.3,10mg/kg/ mice, intravenous, single dose.Treat first latter 48 hours, will, from 3 mice euthanasia of B group, C group and D group, collect as follows their tumor.A tumor is kept in 10% formalin for Histological research.A tumor is freezing for RNA research in liquid nitrogen.A tumor is freezing for the Western trace in liquid nitrogen.The measurement of tumor head carries out twice weekly in 2 weeks, then weekly, continues 4 weeks.When 6 weeks finish or until gross tumor volume reaches about 800-1000mm ³, by all residue mice euthanasia.Dose-response curve shown in first little figure of Figure 32 points out that Apomab 7.3 is that effectively 3mg/kg and 10mg/kg dosage demonstrate obvious (distinct) (although not being statistically significant) improvement with respect to 1mg/kg dosage at all dosage of test.That is compared is all to Tukey-Kramer P<0.05 (seeing second little figure of Figure 32).

Embodiment 7: assess Apomabs23.3,25.3 and 7.3 active anticancer in the Colo205 of human colorectal cancer xenograft models

In this research, give every mice of female nude mouse (Charles River Laboratory) in right drosal part subcutaneous vaccination 0.2ml volume 5 * 10 ⁶individual Colo205 cell.Play the ear label for discriminating to all mices.Allow that tumor reaches mean tumour volume 100-200mm ³, be divided at random seven groups (every group of 10 mices), and treatment as shown in figure 33.The 1st group is medium treatment matched group (10mM histidine, 8% sucrose, 0.02%Tween20 pH6).Use Apomab 7.3 in single dose 3mg/kg/ mouse vein for the 2nd group.Use Apomab 7.3 in single dose 10mg/kg/ mouse vein for the 3rd group.Use Apomab 23.3 in single dose 3mg/kg/ mouse vein for the 4th group.Use Apomab 23.3 in single dose 10mg/kg/ mouse vein for the 5th group.Use Apomab 25.3 in single dose 3mg/kg/ mouse vein for the 6th group.Use Apomab 25.3 in single dose 10mg/kg/ mouse vein for the 7th group.Treat and all mices were weighed in latter 24 hours.The measurement of tumor head carries out twice weekly in 2 weeks, then weekly, continues 4 weeks.6 weeks rear or reach>1000mm of gross tumor volume ³the time, mice is put to death.

The results are shown in Figure 33.As shown in the data of 25 days, Apomab 7.3 and Apomab 25.3 demonstrate significant active anticancer in this model.

Embodiment 8: assessment monoclonal antibody Apomab 7.3 active anticancers for Colo205 human colon carcinoma xenograft in nude mice

animal

Female nude mouse (nu/nu, Harlan) is 11 or 12 week age when the 1st day of research.Arbitrarily feed water and NIH31 Modified and Irradiated Lab Diet to animal

(improvement and irradiation laboratory diet), it contains 18.0% thick protein, 5.0% crude fat, 5.0% crude fibre.Mice is adopt to the irradiated ALPHA-Dri in static micro spacer assembly (static microisolator)

bed-o ' cobs

laboratory Animal Bedding (laboratory animal straw mattress) is upper, illumination in 12 hours circulation, 21-22 ℃, 40-60% humidity.

tumor is implanted

The Colo205 people that xenograft is derived from cultivation contacts cancerous cell.Tumor cell is being supplemented with to 10% heat-inactivated fetal bovine serum, the 100U/mL penicillin G sodium, 100 μ g/mL streptomycin sulfates, 0.25 μ g/mL amphotericin B, 25 μ g/mL gentamycins, the 2mM glutamine, the 1mM Sodium Pyruvate, be cultured to mid-log phase in the RPMI-1640 of 10mM HEPES and 0.075% sodium bicarbonate.In the humidification incubator in 37 ℃ at 5%CO ₂with maintain cell culture in the environment of 95% air in tissue culture flasks.In implantation tumour cell that day, results Colo205 cell, and with 5 * 10 ⁶the concentration of cell/mL is resuspended in the 50%Matrigel substrate (BD Biosciences) in PBS.Every test mice accepts 1 * 10 ⁶individual Colo205 cell, subcutaneous implantation right side, when average-size reaches 100-300mm ³the growing state of time monitoring tumor.After 13 days, be called the 1st day of research, the volume of each tumor is at 126-288mm ³scope in, animal is divided into to six groups, every group by mean tumour volume 188mm ³10 mices form.

test material and treatment

During dosed administration, test material is placed on ice, subsequently dosed administration solution is stored in to 4 ℃.In medium matched group (the 1st group), mice is accepted medium, and intraperitoneal is used, and once a day, continues 5 days, then has a rest 2 days, then once a day, then continues 5 days.In test group (2-4 group), Apo2L.0 part (60mg/kg, intraperitoneal for animal, 5/2/5 timetable), Apomab 7.3 (3mg/kg, intravenous, the 1st and 8 days) and anti-VEGF mouse monoclonal antibody B20-4.1 (10mg/kg, intraperitoneal, the 1st and 8 days).The 5th and 6 groups accept respectively the combination of B20-4.1 and Apo2L.0 and the combination of B20-4.1 and Apomab 7.3.Every dose of volume with the every 20g body weight of 0.2mL (10mL/kg) is delivered, and according to the weight of animals, determines in proportion.

terminal

Use slide calliper rule (caliper) to measure tumor twice weekly.When the gross tumor volume of animal reaches 2000mm ³perhaps, when within the 68th day, finishing research, get and first send out the survivor, by every animal euthanasia.Owing to not reaching 2000mm ³the tumor number of size, for 1000mm is selected in tumor growth delay research ³terminal (endpoint) gross tumor volume.Calculated the time to terminal (TTE) of every mice by following equation:

TTE (my god)=[log ₁₀(terminal volume, mm ³)-b]/m

Wherein b and m are respectively intercept and the slopes of the straight line that obtains of the linear regression of the log value by transforming the tumor growth data set.This data set comprises observed result for the first time and proper three the Continuous Observation results before the terminal volume that surpass research terminal volume.The animal that does not reach terminal is assigned to the TTE value, equal the last day of research.To being treated associated death or assigning the TTE value because transfer suffers the animal of non-treatment associated death, equal dead that day.Got rid of outside TTE calculates by the animal of the death of non-treatment associated death or reason the unknown.

The treatment achievement is assessed by tumor growth delay (TGD), and it is defined as treatment group and compares the intermediate value growth of time (TTE) to terminal with matched group:

TGD＝T-C，

In sky, or state the percentage ratio with respect to matched group intermediate value TTE as:

％TGD＝(T-C)/C×100，

Wherein

The intermediate value TTE of T=treatment group,

The intermediate value TTE of C=matched group.

Treatment can cause that in animal, the part of tumor disappears (PR) or disappear fully (CR).In PR response, the continuous gross tumor volume of measuring for three times during research be its 1st day volume 50% or below, and the one or many in measuring for these three times is equal to or greater than 13.5mm ³.In the CR response, the one or many gross tumor volume in three measurements of this during research is less than 13.5mm ³.The animal that has the CR response when research finishes is included in addition without tumor survival person (TFS).Monitor and record the response (Regressionresponse) of disappearing.

sampling

Animal when terminal from every group gathers tumor sample.Just before sampling by cervical dislocation by these animal euthanasias.Gather the tumor of every group of three animals, cut in half, and be stored in the formalin of 10% neutral buffered room temperature 12-24 hour.

statistics and pattern analysis

Check to analyze the significance of difference between treatment group and matched group TTE value with Logrank.Significance level at P=0.05 carries out two tail statistical analysis (two-tailed statistical analysis), thinks that the result of 0.01≤P≤0.05 is significant, and the result of P<0.01 is highly significant.

The intermediate value tumor growth curve is shown as midvalue of class gross tumor volume (group median tumor volume) function of time.When animal, because tumor size exits when research, will be included in for the final gross tumor volume of this animal record in the data for the midvalue of class gross tumor volume of calculated for subsequent time point.Draw the Kaplan-Meier curve chart and remain the function that the percentages show of animal is the time in study.These curve charts are used and check identical data set with Logrank.

result

Figure 34 has shown in this research midvalue of class tumor growth curve (upper figure) and the Kaplan-Meier curve chart (figure below) of every group.The intermediate value TTE of medium treatment control mice is 10.0 days, and wherein 1 tumor (10 altogether) does not reach 1000mm ³the terminal gross tumor volume.The 1st with use B20-4.1 with the 10mg/kg intraperitoneal in 8 days, produce 19.9 days (113%) TGD of appropriateness, it is inapparent statistically.Apo2L.0 and Apomab 7.3 monotherapies are effectively to Colo205, produce respectively the tumor growth delay of 28.4 days (190%) and 53.0 days (355%).Arbitrary interpolation B20-4.1 in Apo2L.0 or Apomab 7.3 is not being improved to therapeutic efficiency aspect TGD or the response of disappearing.

Embodiment 9: assessment adds the active anticancer of the monoclonal antibody Apomab7.3 of paclitaxel for SKMES-1 people NSCLC as the associating carboplatin that reaches of monotherapy

Test as monotherapy and the associating carboplatin add the Apo2L.0 part of paclitaxel (paclitaxel) and the Apomab 7.3 anti-tumor in vivo activity for SKMES-1 people NSCLC.

animal

Female nude mouse (nu/nu, Harlan) is 9-10 age in week when the 1st day of research, body weight 17.4-25.4g.Arbitrarily feed water and NIH31 Modified and Irradiated Lab Diet to animal

(improvement and irradiation laboratory diet), it contains 18.0% thick protein, 5.0% crude fat, 5.0% crude fibre.Mice is adopt to the irradiatedALPHA-Dri in static micro spacer assembly (static microisolator)

bed-o ' cobs

laboratory Animal Bedding (laboratory animal straw mattress) is upper, illumination in 12 hours circulation, 21-22 ℃, 40-60% humidity.

tumor is implanted

Xenograft is derived from the SKMES-1 lung tumor of cultivation, and it is by transplanting and maintain continuously at PRC.Every test mice is accepted 1mm ³the SKMES-1 tumor fragment, subcutaneous implantation right side, and the growing state of monitoring tumor.After 13 days, be called the 1st day of research, the volume of each tumor is at 63-144mm ³scope in, animal is divided into to six groups, wherein four groups each by 10 mices, formed, two groups each by 9 mices, formed.Mean tumour volume is 93-95mm ³.

test material and treatment

It is ready as dosed administration that all test materials be take the every 20g body weight of 0.1mL (5mL/kg), is stored in-80 ℃ when receiving.First day at dosed administration melts test material, during dosed administration, is placed on ice, is stored in subsequently 4 ℃.Carboplatin (PARAPLATIN

injection, Bristol MyersSquibb) 5% dextrose dilution in water (D5W), to produce the desired amount of every 10g body weight (10mL/kg) 0.2mL volume.Paclitaxel (Natural Pharmaceuticals, Inc.) in every day of dosed administration, with D5W, from the 10X liquid storage, dilute, to produce medium, its 5% ethanol and 5% cremophor (Cremophor) EL in 90%D5W forms (5%EC medium), makes desired amount deliver at every 20g body weight 0.1mL.

The 1st group of (medium contrast (vehicle control)) mice accepted medium, and intraperitoneal is used, and once a day, continues 5 days (i.p.qdx5), serves as the tumor growth contrast.The 2nd and the mice of 3 groups accept respectively Apo2L.0 (60mg/kg, subcutaneous, qdx5) and Apomab 7.3 (10mg/kg, intravenous, monotherapy qdx1).The mice of the 4th group accept carboplatin (100mg/kg, intraperitoneal, qdx1) add paclitaxel (6.25mg/kg, subcutaneous, qdx5) combination.The 5th and the mice of 6 groups accept respectively the combination that carboplatin adds paclitaxel and Apo2L or Apomab 7.3.Before every potion, the described volume of a joint is used, and according to the weight of animals, determines in proportion.

terminal, sampling and statistical analysis

Determine terminal described in front embodiment 8, sampled and statistical analysis.

result

Figure 35 and 36 has shown respectively midvalue of class tumor growth curve and the Kaplan-Meier curve chart by the group of Apo2L.0 and Apomab 7.3 treatments.

The tumor growth of all medium treatment control mice is to 1500mm ³the terminal volume, intermediate value TTE is 18.9 days.Therefore, in research in these 45 days, attainable maximum TGD is 26.1 days (138%).The intermediate value tumor growth curve of matched group and Kaplan-Meier curve chart are included in the upper figure and figure below of Figure 35 and 36.

For Apo2L.0 treatment group (the 2nd group), intermediate value TTE is 22.9 days, corresponding to 4.0 days (21%) TGD and the inapparent activity of statistics.Figure 28 (upper figure) points out that the intermediate value gross tumor volume of the 2nd group dwindles during treating, and then tumor growth recovers fast.

The intermediate value TTE of the 3rd group is 2.0 days, corresponding to the activity (P=0.005) of 7.1 days (38%) TGD and statistics highly significant.The record that does not disappear and respond, all tumors reach 1500mm ³the terminal volume.The intermediate value tumor growth curve of the 3rd group demonstrates the initial delay of tumor growth with respect to contrast.

The intermediate value TTE that adds the 4th group of mice of paclitaxel treatment with carboplatin is 26.5 days, corresponding to the activity (P=0.01) of 7.6 days (40%) TGF and statistically significant.All tumor growths of the 4th group are to 1500mm ³the terminal volume, the record of the response of not disappearing.The intermediate value tumor growth curve of the 4th group of mice is pointed out the moderate retardation (modest delay) (see Figure 35 and 36, upper figure) of tumor growth with respect to control mice.

Treatment that Apo2L.0, carboplatin and paclitaxel three joint groups close produces the intermediate value TTE of 32.7 days, corresponding to 13.9 days (73%) TGD with respect to the activity (P=0.002) of the 1st group of statistics highly significant.32.7 days intermediate value TTE that this three joint group closes are longer than 16.5 days intermediate value TTE of 22.9 days intermediate value TTE of Apo2L.0 monotherapy group or chemotherapy matched group, but analyze according to Logrank, and this difference does not reach significance,statistical.Although the shortage significance,statistical, the intermediate value tumor growth curve points out that the therapeutic alliance of the 5th group adds Paclitaxel Chemotherapy with Apo2L.0 monotherapy or carboplatin and compared greater activity (seeing Figure 28, upper figure).

The treatment that three joint groups of Apomab 7.3, carboplatin and paclitaxel close (the 6th group) causes 3/10 TR death, and therefore, this group can not be assessed TGD.Yet the intermediate value tumor growth curve points out that the therapeutic alliance of the 6th group adds Paclitaxel Chemotherapy with Apomab 7.3 monotherapies or carboplatin and compared greater activity (seeing Figure 37, upper figure).

conclusion

Although this experiment has than high mortality, arbitraryly in data declaration Apo2L.0 and Apomab 7.3 can increase antitumous effect to the treatment of carboplatin and paclitaxel.

Embodiment 10: in people Colo205 cancer xenograft models, assessment is as active anticancer monotherapy and the monoclonal antibody Apomab 7.3 associating VEGF antibody

animal

Female nude mouse (nu/nu, Harlan) is 7-8 age in week when the 1st day of research.Arbitrarily feed water and NIH31 Modified and Irradiated Lab Diet to animal

(improvement and irradiation laboratory diet), it contains 18.0% thick protein, 5.0% crude fat, 5.0% crude fibre.Mice is adopt to the irradiated ALPHA-Dri in static micro spacer assembly (static microisolator)

bed-o ' cobs

laboratory Animal Bedding (laboratory animal straw mattress) is upper, illumination in 12 hours circulation, 21-22 ℃, 40-60% humidity.

tumor cell culture

People Colo205 colon cancer cell is containing the 100U/mL penicillin G sodium, and 100 μ g/mL streptomycin sulfates are cultivated in the RPMI1640 culture medium of 0.25 μ g/mL amphotericin B and 25 μ g/mL gentamycins.Culture media supplemented has 10% heat-inactivated fetal bovine serum, 2mM glutamine and 1mM sodium bicarbonate.Tumor cell in tissue culture flasks in the humidification incubator in 37 ℃ at 5%CO ₂with in the environment of 95% air, cultivate.

body is implanted into

The people Colo205 cancerous cell of results for implanting in the logarithmic (log) phase growth course, and with 5 * 10 ⁶cell/mL is resuspended in 50%Matrigel.Every mice is at right side subcutaneous injection 1 * 10 ⁶individual cell (0.2mL cell suspending liquid).Twice monitoring tumor weekly, when their volume reaches 100-300mm ³the time every day monitoring once.Research the 1st day, animal is divided into to treatment group, gross tumor volume 108.0-220.5mm ³, group mean tumour volume 149.8mm ³.Can estimate tumor weight, 1mg is equivalent to 1mm ³gross tumor volume.

test material and treatment

Provide test material to carry out dosed administration.Dosed administration solution is stored in to 4 ℃.

Mice is divided into to six groups, every group of ten mices.All treatments are all that intraperitoneal (i.p.) is used.

Apo2L.0 and medium thereof are used once (qdx5) each comfortable 1-5 days every days.Apomab 7.3 and the anti-G6 of mouse-anti VEGF antibody (anti-G6) are respectively at the 1st day be administered once (qdx1).Contrast the medium treatment of the 1st group of mice with Apo2L.0.Accept Apo2L.0 monotherapy, 60mg/kg for the 2nd group.Accept Apomab 7.3 monotherapies, 3mg/kg for the 3rd group.Accept BY4 monotherapy, 5mg/kg for the 4th group.The 5th and 6 groups accept respectively the Apo2L.0 of 60mg/kg and the Apomab 7.3 of 3mg/kg, each combines the anti-G6 of 5mg/kg.In all groups, the dosed administration volume of 0.2mL/20g mice determines in proportion according to the body weight of every animal.

terminal, sampling and statistical analysis

Determine terminal described in front embodiment 8, sampled and statistical analysis.

result

The results are shown in Figure 37, wherein the curve display midvalue of class gross tumor volume in upper figure is with respect to the diagram of time, and the Kaplan-Meier curve chart in figure below shows that respectively the group residue can be assessed the diagram of animal percentage ratio with respect to the time.

Contrast the 1st group of mice and accept the Apo2L.0 medium, serve as the contrast of all treatment groups.Tumor growth in all 10 mices is to 1500mm ³the terminal volume, intermediate value TTE is 20.8 days.Therefore, the maximum possible TGD in research in these 61 days is 193%.

Accept Apo2L.0 monotherapy, 60mg/kg for the 2nd group.This treatment produces the anti-tumor activity (P<0.001) of highly significant with respect to the medium matched group, intermediate value TTE is 53.6 days.This intermediate value TTE is corresponding to 32.8 days T-C and 158%TGD.5 mices are 1,210mm at the intermediate value gross tumor volume of the 61st day ³.Recorded 1 routine LTTFS.

Accept Apomab 7.3 monotherapies, 3mg/kg for the 3rd group.This treatment produces the activity (P<0.001) of highly significant, and maximum possible TGD is that 193%, MTV (6) is 776mm ³.Be recorded to 1 routine LTTFS, 1 routine instantaneous CR response and 3 routine FR responses.

Accept anti-G6 monotherapy, 5mg/kg for the 4th group.This treatment produces the activity (P<0.01) of highly significant, and TGF is that 86%, MTV (3) is 1,224mm ³.Be not recorded to the response of disappearing.

The combination of the anti-G6 of the 5th group of Apo2L.0 that accepts 60mg/kg and 5mg/kg.This therapeutic alliance produces 138%TGD.Anti-tumor activity is treated highly significant (P<0.001) with respect to medium, but not remarkable with respect to two kinds of monotherapies.In the 5th group, MTV (3) is 1,080mm ³, be recorded to 1 routine PR response.

The therapeutic alliance of the anti-G6 of the 6th group of Apomab 7.3 that accepts 3mg/kg and 5mg/kg.This treatment produces 193% maximum possible TGD.Anti-tumor activity is (P<0.001) of highly significant with respect to the medium treatment, is significant with respect to anti-G6 monotherapy, but is inapparent with respect to Apomab 7.3 monotherapies.In the 6th group, MTV (8) is 208mm ³, be recorded to 5 routine PR responses.

conclusion

Tumor has strong response to the monotherapy (the 3rd group) of 3mg/kg qdx1 Apomab 7.3.This treatment produces the activity of highly significant with respect to the medium matched group, maximum possible TGD is 193%.In survival to the 61st day, MTV, be 776mm ³6 mices in, tumor regression has occurred in 5 animals.This monotherapy produces 1 routine LTTFS, 1 routine instantaneous CR response and 3 routine PR responses.The intermediate value gross tumor volume is until just increase (Figure 37) after the 15th day.

The conjoint therapy of Apomab 7.3 and the anti-G6 of mouse-anti VEGF antibody produces independent Apomab 7.3 or the stronger activity of the viewed activity of anti-G6 for ratio.This therapeutic alliance produces 8 61 days survivors, has produced the minimum MTV of research, is 208mm ³.The intermediate value tumor growth curve shows that tumor dwindles or stagnate until the 33rd day, and tumor growth subsequently is (Figure 37) very slowly also.This therapeutic alliance produces than the remarkable stronger activity of anti-G6 monotherapy, but there is no significant difference with the result of Apomab 7.3 monotherapies.In addition, this therapeutic alliance produces 5 routine PR responses, and 5 examples that obtain with Apomab 7.3 monotherapies disappear to respond and comprise 1 routine instantaneous CR and 1 routine LTTFS.

All therapies are all well tolerated.Do not observe under study for action and lose weight or other overt toxicity.

In a word, the anti-G6 conjoint therapy of Apomab 7.3/ produces more 61 days survivors and lower MTV than the treatment of corresponding monotherapy.Yet the anti-G6 associating of Apomab 7.3/ does not produce cures active (curative activity), and this has observed in Apomab 7.3 monotherapies.

Sequence table

<110 > Genentech Inc (GENENTECH, INC.)

ADAMS，CAMELLI A W.

<120 > DR5 antibody and uses thereof

<130>39766-0164

<140 > to be specified

<141 > herewith

<150>US 60/649,550

<151>2005-02-02

<160>29

<170>Fa stSEQ for Windows Ver s ion 4.0

<210>1

<211>281

<212>PRT

<213 > mankind (Homo sapiens)

<400>1

Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys

1 5 10 15

Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala

20 25 30

Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys

35 40 45

Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr

50 55 60

Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val

65 70 75 80

Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser

85 90 95

Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro

100 105 110

Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly

115 120 125

Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu

130 135 140

Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly

145 150 155 160

His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile

165 170 175

His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe

180 185 190

Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln

195 200 205

Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys

210 215 220

Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr

225 230 235 240

Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile

245 250 255

Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala

260 265 270

Ser Phe Phe Gly Ala Phe Leu Val Gly

275 280

<210>2

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tttcctcact gactataaaa gaatagagaa ggaagggctt cagtgaccgg ctgcctggct 60

gacttacagc agtcagactc tgacaggatc atggctatga tggaggtcca ggggggaccc 120

agcctgggac agacctgcgt gctgatcgtg atcttcacag tgctcctgca gtctctctgt 180

gtggctgtaa cttacgtgta ctttaccaac gagctgaagc agatgcagga caagtactcc 240

aaaagtggca ttgcttgttt cttaaaagaa gatgacagtt attgggaccc caatgacgaa 300

gagagtatga acagcccctg ctggcaagtc aagtggcaac tccgtcagct cgttagaaag 360

atgattttga gaacctctga ggaaaccatt tctacagttc aagaaaagca acaaaatatt 420

tctcccctag tgagagaaag aggtccncag agagtagcag ctcacataac tgggaccaga 480

ggaagaagca acacattgtc ttctccaaac tccaagaatg aaaaggctct gggccgcaaa 540

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aatggtgaac tggtcatcca tgaaaaaggg ttttactaca tctattccca aacatacttt 660

cgatttcagg aggaaataaa agaaaacaca aagaacgaca aacaaatggt ccaatatatt 720

tacaaataca caagttatcc tgaccctata ttgttgatga aaagtgctag aaatagttgt 780

tggtctaaag atgcagaata tggactctat tccatctatc aagggggaat atttgagctt 840

aaggaaaatg acagaatttt tgtttctgta acaaatgagc acttgataga catggaccat 900

gaagccagtt ttttcggggc ctttttagtt ggctaactga cctggaaaga aaaagcaata 960

acctcaaagt gactattcag ttttcaggat gatacactat gaagatgttt caaaaaatct 1020

gaccaaaaca aacaaacaga aa 1042

<210>3

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<212>PRT

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Met Ala Pro Pro Pro Ala Arg Val His Leu Gly Ala Phe Leu Ala Val

1 5 10 15

Thr Pro Asn Pro Gly Ser Ala Ala Ser Gly Thr Glu Ala Ala Ala Ala

20 25 30

Thr Pro Ser Lys Val Trp Gly Ser Ser Ala Gly Arg Ile Glu Pro Arg

35 40 45

Gly Gly Gly Arg Gly Ala Leu Pro Thr Ser Met Gly Gln His Gly Pro

50 55 60

Ser Ala Arg Ala Arg Ala Gly Arg Ala Pro Gly Pro Arg Pro Ala Arg

65 70 75 80

Glu Ala Ser Pro Arg Leu Arg Val His Lys Thr Phe Lys Phe Val Val

85 90 95

Val Gly Val Leu Leu Gln Val Val Pro Ser Ser Ala Ala Thr Ile Lys

100 105 110

Leu His Asp Gln Ser Ile Gly Thr Gln Gln Trp Glu His Ser Pro Leu

115 120 125

Gly Glu Leu Cys Pro Pro Gly Ser His Arg Ser Glu Arg Pro Gly Ala

130 135 140

Cys Asn Arg Cys Thr Glu Gly Val Gly Tyr Thr Asn Ala Ser Asn Asn

145 150 155 160

Leu Phe Ala Cys Leu Pro Cys Thr Ala Cys Lys Ser Asp Glu Glu Glu

165 170 175

Arg Ser Pro Cys Thr Thr Thr Arg Asn Thr Ala Cys Gln Cys Lys Pro

180 185 190

Gly Thr Phe Arg Asn Asp Asn Ser Ala Glu Met Cys Arg Lys Cys Ser

195 200 205

Thr Gly Cys Pro Arg Gly Met Val Lys Val Lys Asp Cys Thr Pro Trp

210 215 220

Ser Asp Ile Glu Cys Val His Lys Glu Ser Gly Asn Gly His Asn Ile

225 230 235 240

Trp Val Ile Leu Val Val Thr Leu Val Val Pro Leu Leu Leu Val Ala

245 250 255

Val Leu Ile Val Cys Cys Cys Ile Gly Ser Gly Cys Gly Gly Asp Pro

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Lys Cys Met Asp Arg Val Cys Phe Trp Arg Leu Gly Leu Leu Arg Gly

275 280 285

Pro Gly Ala Glu Asp Asn Ala His Asn Glu Ile Leu Ser Asn Ala Asp

290 295 300

Ser Leu Ser Thr Phe Val Ser Glu Gln Gln Met Glu Ser Gln Glu Pro

305 310 315 320

Ala Asp Leu Thr Gly Val Thr Val Gln Ser Pro Gly Glu Ala Gln Cys

325 330 335

Leu Leu Gly Pro Ala Glu Ala Glu Gly Ser Gln Arg Arg Arg Leu Leu

340 345 350

Val Pro Ala Asn Gly Ala Asp Pro Thr Glu Thr Leu Met Leu Phe Phe

355 360 365

Asp Lys Phe Ala Asn Ile Val Pro Phe Asp Ser Trp Asp Gln Leu Met

370 375 380

Arg Gln Leu Asp Leu Thr Lys Asn Glu Ile Asp Val Val Arg Ala Gly

385 390 395 400

Thr Ala Gly Pro Gly Asp Ala Leu Tyr Ala Met Leu Met Lys Trp Val

405 410 415

Asn Lys Thr Gly Arg Asn Ala Ser Ile His Thr Leu Leu Asp Ala Leu

420 425 430

Glu Arg Met Glu Glu Arg His Ala Lys Glu Lys Ile Gln Asp Leu Leu

435 440 445

Val Asp Ser Gly Lys Phe Ile Tyr Leu Glu Asp Gly Thr Phe Ser Ala

450 455 460

Val Ser Leu Glu

465

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agcaaagtgt ggggctcttc cgcggggagg attgaaccac gaggcggggg ccgaggagcg 180

ctccctacct ccatgggaca gcacggaccc agtgcccggg cccgggcagg gcgcgcccca 240

ggacccaggc cggcgcggga agccagccct cggctccggg tccacaagac cttcaagttt 300

gtcgtcgtcg gggtcctgct gcaggtcgta cctagctcag ctgcaaccat caaacttcat 360

gatcaatcaa ttggcacaca gcaatgggaa catagccctt tgggagagtt gtgtccacca 420

ggatctcata gatcagaacg tcctggagcc tgtaaccggt gcacagaggg tgtgggttac 480

accaatgctt ccaacaattt gtttgcttgc ctcccatgta cagcttgtaa atcagatgaa 540

gaagagagaa gtccctgcac cacgaccagg aacacagcat gtcagtgcaa accaggaact 600

ttccggaatg acaattctgc tgagatgtgc cggaagtgca gcacagggtg ccccagaggg 660

atggtcaagg tcaaggattg tacgccctgg agtgacatcg agtgtgtcca caaagaatca 720

ggcaatggac ataatatatg ggtgattttg gttgtgactt tggttgttcc gttgctgttg 780

gtggctgtgc tgattgtctg ttgttgcatc ggctcaggtt gtggagggga ccccaagtgc 840

atggacaggg tgtgtttctg gcgcttgggt ctcctacgag ggcctggggc tgaggacaat 900

gctcacaacg agattctgag caacgcagac tcgctgtcca ctttcgtctc tgagcagcaa 960

atggaaagcc aggagccggc agatttgaca ggtgtcactg tacagtcccc aggggaggca 1020

cagtgtctgc tgggaccggc agaagctgaa gggtctcaga ggaggaggct gctggttcca 1080

gcaaatggtg ctgaccccac tgagactctg atgctgttct ttgacaagtt tgcaaacatc 1140

gtgccctttg actcctggga ccagctcatg aggcagctgg acctcacgaa aaatgagatc 1200

gatgtggtca gagctggtac agcaggccca ggggatgcct tgtatgcaat gctgatgaaa 1260

tgggtcaaca aaactggacg gaacgcctcg atccacaccc tgctggatgc cttggagagg 1320

atggaagaga gacatgcaaa agagaagatt caggacctct tggtggactc tggaaagttc 1380

atctacttag aagatggcac aggctctgcc gtgtccttgg agtgaaagac tctttttacc 1440

agaggtttcc tcttaggtgt taggagttaa tacatattag gttttttttt tttttaacat 1500

gtatacaaag taaattctta gccacgtgta ttggctcctg cctgtaatcc catcactttg 1560

ggaggctgac gccggtggat ccacttgagg tccgaagttc caagaccagc cctgaaccaa 1620

catcgtggaa atgcccgtct tttacaaaaa aataccaaaa attcaactgg aatgtgcatg 1680

gtgtgtgcca tcatttcctc ggctaactac gggaggtctg aggccaggag aatccacttg 1740

aaccccacga aggacagtgt agactgcaga ttgcaccact gcactcccag cctgggaaca 1800

cagagcaaga ctctgtctca agataaaata aaataaactt gaaagaatta ttgcccgact 1860

gaggctcaca tgccaaagga aaatctggtt ctcccctgag ctggcctccg tgtgtttcct 1920

tatcatggtg gtcaattgga ggtgttaatt tgaatggatt aaggaacacc tagaacactg 1980

gtaaggcatt atttctggga cattatttct gggcatgtct tcgagggtgt ttccagaggg 2040

gattggcatg cgatcgggtg gactgagtgg aaaagaccta cccttaattt gggggggcac 2100

cgtccgacag actggggagc aagatagaag aaaacaaaaa aaaaaaaaaa aa 2152

<210>5

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<223 > Xaa=Leu or Met

<400>5

Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys

1 5 10 15

Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu

20 25 30

Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu

35 40 45

Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln

50 55 60

Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu

65 70 75 80

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

85 90 95

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

100 105 110

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

115 120 125

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

130 135 140

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

145 150 155 160

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

165 170 175

Glu Cys Val His Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala

180 185 190

Ala Val Val Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp

195 200 205

Lys Lys Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly

210 215 220

Asp Pro Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp

225 230 235 240

Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro

245 250 255

Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn

260 265 270

Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala

275 280 285

Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp

290 295 300

Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val

305 310 315 320

Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp

325 330 335

Asn Glu Ile Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr

340 345 350

Leu Tyr Thr Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala

355 360 365

Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu

370 375 380

Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met

385 390 395 400

Tyr Leu Glu Gly Asn Ala Asp Ser Ala Xaa Ser

405 410

<210>6

<211>1799

<212>DNA

<213 > mankind

<400>6

cccacgcgtc cgcataaatc agcacgcggc cggagaaccc cgcaatctct gcgcccacaa 60

aatacaccga cgatgcccga tctactttaa gggctgaaac ccacgggcct gagagactat 120

aagagcgttc cctaccgcca tggaacaacg gggacagaac gccccggccg cttcgggggc 180

ccggaaaagg cacggcccag gacccaggga ggcgcgggga gccaggcctg ggctccgggt 240

ccccaagacc cttgtgctcg ttgtcgccgc ggtcctgctg ttggtctcag ctgagtctgc 300

tctgatcacc caacaagacc tagctcccca gcagagagcg gccccacaac aaaagaggtc 360

cagcccctca gagggattgt gtccacctgg acaccatatc tcagaagacg gtagagattg 420

catctcctgc aaatatggac aggactatag cactcactgg aatgacctcc ttttctgctt 480

gcgctgcacc aggtgtgatt caggtgaagt ggagctaagt ccctgcacca cgaccagaaa 540

cacagtgtgt cagtgcgaag aaggcacctt ccgggaagaa gattctcctg agatgtgccg 600

gaagtgccgc acagggtgtc ccagagggat ggtcaaggtc ggtgattgta caccctggag 660

tgacatcgaa tgtgtccaca aagaatcagg catcatcata ggagtcacag ttgcagccgt 720

agtcttgatt gtggctgtgt ttgtttgcaa gtctttactg tggaagaaag tccttcctta 780

cctgaaaggc atctgctcag gtggtggtgg ggaccctgag cgtgtggaca gaagctcaca 840

acgacctggg gctgaggaca atgtcctcaa tgagatcgtg agtatcttgc agcccaccca 900

ggtccctgag caggaaatgg aagtccagga gccagcagag ccaacaggtg tcaacatgtt 960

gtcccccggg gagtcagagc atctgctgga accggcagaa gctgaaaggt ctcagaggag 1020

gaggctgctg gttccagcaa atgaaggtga tcccactgag actctgagac agtgcttcga 1080

tgactttgca gacttggtgc cctttgactc ctgggagccg ctcatgagga agttgggcct 1140

catggacaat gagataaagg tggctaaagc tgaggcagcg ggccacaggg acaccttgta 1200

cacgatgctg ataaagtggg tcaacaaaac cgggcgagat gcctctgtcc acaccctgct 1260

ggatgccttg gagacgctgg gagagagact tgccaagcag aagattgagg accacttgtt 1320

gagctctgga aagttcatgt atctagaagg taatgcagac tctgccwtgt cctaagtgtg 1380

attctcttca ggaagtgaga ccttccctgg tttacctttt ttctggaaaa agcccaactg 1440

gactccagtc agtaggaaag tgccacaatt gtcacatgac cggtactgga agaaactctc 1500

ccatccaaca tcacccagtg gatggaacat cctgtaactt ttcactgcac ttggcattat 1560

ttttataagc tgaatgtgat aataaggaca ctatggaaat gtctggatca ttccgtttgt 1620

gcgtactttg agatttggtt tgggatgtca ttgttttcac agcacttttt tatcctaatg 1680

taaatgcttt atttatttat ttgggctaca ttgtaagatc catctacaaa aaaaaaaaaa 1740

aaaaaaaaag ggcggccgcg actctagagt cgacctgcag aagcttggcc gccatggcc 1799

<210>7

<211>440

<212>PRT

<213 > mankind

<400>7

Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys

1 5 10 15

Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro

20 25 30

Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu

35 40 45

Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln

50 55 60

Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu

65 70 75 80

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

85 90 95

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

100 105 110

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

115 120 125

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

130 135 140

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

145 150 155 160

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

165 170 175

Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro

180 185 190

Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro

195 200 205

Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val

210 215 220

Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val

225 230 235 240

Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu

245 250 255

Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu

260 265 270

Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu

275 280 285

Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser

290 295 300

Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser

305 310 315 320

Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu

325 330 335

Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp

340 345 350

Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu Ile

355 360 365

Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr

370 375 380

Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His

385 390 395 400

Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln

405 410 415

Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu

420 425 430

Gly Asn Ala Asp Ser Ala Met Ser

435 440

<210>8

<211>1323

<212>DNA

<213 > mankind

<400>8

atggaacaac ggggacagaa cgccccggcc gcttcggggg cccggaaaag gcacggccca 60

ggacccaggg aggcgcgggg agccaggcct gggctccggg tccccaagac ccttgtgctc 120

gttgtcgccg cggtcctgct gttggtctca gctgagtctg ctctgatcac ccaacaagac 180

ctagctcccc agcagagagt ggccccacaa caaaagaggt ccagcccctc agagggattg 240

tgtccacctg gacaccatat ctcagaagac ggtagagatt gcatctcctg caaatatgga 300

caggactata gcactcactg gaatgacctc cttttctgct tgcgctgcac caggtgtgat 360

tcaggtgaag tggagctaag tccctgcacc acgaccagaa acacagtgtg tcagtgcgaa 420

gaaggcacct tccgggaaga agattctcct gagatgtgcc ggaagtgccg cacagggtgt 480

cccagaggga tggtcaaggt cggtgattgt acaccctgga gtgacatcga atgtgtccac 540

aaagaatcag gtacaaagca cagtggggaa gccccagctg tggaggagac ggtgacctcc 600

agcccaggga ctcctgcctc tccctgttct ctctcaggca tcatcatagg agtcacagtt 660

gcagccgtag tcttgattgt ggctgtgttt gtttgcaagt ctttactgtg gaagaaagtc 720

cttccttacc tgaaaggcat ctgctcaggt ggtggtgggg accctgagcg tgtggacaga 780

agctcacaac gacctggggc tgaggacaat gtcctcaatg agatcgtgag tatcttgcag 840

cccacccagg tccctgagca ggaaatggaa gtccaggagc cagcagagcc aacaggtgtc 900

aacatgttgt cccccgggga gtcagagcat ctgctggaac cggcagaagc tgaaaggtct 960

cagaggagga ggctgctggt tccagcaaat gaaggtgatc ccactgagac tctgagacag 1020

tgcttcgatg actttgcaga cttggtgccc tttgactcct gggagccgct catgaggaag 1080

ttgggcctca tggacaatga gataaaggtg gctaaagctg aggcagcggg ccacagggac 1140

accttgtaca cgatgctgat aaagtgggtc aacaaaaccg ggcgagatgc ctctgtccac 1200

accctgctgg atgccttgga gacgctggga gagagacttg ccaagcagaa gattgaggac 1260

cacttgttga gctctggaaa gttcatgtat ctagaaggta atgcagactc tgccatgtcc 1320

taa 1323

<210>9

<211>930

<212>DNA

<213 > mankind

<400>9

atgaccatga ttacgccaag ctttggagcc ttttttttgg agattttcaa cgtgaaaaaa 60

ttattattcg caattccttt agttgttcct ttctatgcgg cccagccggc catggccgag 120

gtgcagctgg tgcagtctgg gggaggtgtg gaacggccgg gggggtccct gagactctcc 180

tgtgcagcct ctggattcac ctttgatgat tatggcatga gctgggtccg ccaagctcca 240

gggaaggggc tggagtgggt ctctggtatt aattggaatg gtggtagcac aggatatgca 300

gactctgtga agggccgagt caccatctcc agagacaacg ccaagaactc cctgtatctg 360

caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgcgaa aatcctgggt 420

gccggacggg gctggtactt cgatctctgg gggaagggga ccacggtcac cgtctcgagt 480

ggtggaggcg gttcaggcgg aggtggcagc ggcggtggcg gatcgtctga gctgactcag 540

gaccctgctg tgtctgtggc cttgggacag acagtcagga tcacatgcca aggagacagc 600

ctcagaagct attatgcaag ctggtaccag cagaagccag gacaggcccc tgtacttgtc 660

atctatggta aaaacaaccg gccctcaggg atcccagacc gattctctgg ctccagctca 720

ggaaacacag cttccttgac catcactggg gctcaggcgg aagatgaggc tgactattac 780

tgtaactccc gggacagcag tggtaaccat gtggtattcg gcggagggac caagctgacc 840

gtcctaggtg cggccgcaca tcatcatcac catcacgggg ccgcagaaca aaaactcatc 900

tcagaagagg atctgaatgg ggccgcatag 930

<210>10

<211>309

<212>PRT

<213 > mankind

<400>10

Met Thr Met Ile Thr Pro Ser Phe Gly Ala Phe Phe Leu Glu Ile Phe

1 5 10 15

Asn Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr

20 25 30

Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Gln Ser Gly Gly

35 40 45

Gly Val Glu Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

50 55 60

Gly Phe Thr Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro

65 70 75 80

Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser

85 90 95

Thr Gly Tyr Ala Asp Ser Val Lys Gly Arg Val Thr Ile Ser Arg Asp

100 105 110

Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

115 120 125

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Ile Leu Gly Ala Gly Arg Gly

130 135 140

Trp Tyr Phe Asp Leu Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

165 170 175

Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val

180 185 190

Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp

195 200 205

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys

210 215 220

Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

225 230 235 240

Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu

245 250 255

Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val Val

260 265 270

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ala Ala Ala His His

275 280 285

His His His His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp

290 295 300

Leu Asn Gly Ala Ala

305

<210>11

<211>451

<212>PRT

<213 > mankind

<400>11

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ile Leu Gly Ala Gly Arg Gly Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>12

<211>1353

<212>DNA

<213 > mankind

<400>12

gaagttcagc tggtgcagtc tgggggaggt gtggaacggc cgggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120

ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggatat 180

gcagactctg tgaagggccg agtcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaatcctg 300

ggtgccggac ggggctggta cttcgatctc tgggggaagg ggaccacggt caccgtctcg 360

agtgcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtggtg actgtgccct ctagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggaa 1080

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccgggt aaa 1353

<210>13

<211>213

<212>PRT

<213 > mankind

<400>13

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr

1 5 10 15

Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly

35 40 45

Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser

50 55 60

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys Ser

210

<210>14

<211>639

<212>DNA

<213 > mankind

<400>14

tctgagctga ctcaggaccc tgctgtgtct gtggccttgg gacagacagt caggatcaca 60

tgccaaggag acagcctcag aagctattat gcaagctggt accagcagaa gccaggacag 120

gcccctgtac ttgtcatcta tggtaaaaac aaccggccct cagggatccc agaccgattc 180

tctggctcca gctcaggaaa cacagcttcc ttgaccatca ctggggctca ggcggaagat 240

gaggctgact attactgtaa ctcccgggac agcagtggta accatgtggt attcggcgga 300

gggaccaagc tgaccgtcct tggccaacct aaggctgcac catctgtcac cctcttcccg 360

ccatcttctg aggagttgca agctaacaaa gccactcttg tgtgcctgat cagtgacttc 420

tatcccggag cggtcacagt agcgtggaag gcggatagct cccccgtaaa ggctggcgtc 480

gagacgacta ccccttcgaa gcagagcaac aacaaatacg ccgccagcag ctacctgtcg 540

ctgaccccag aacagtggaa gagccacaaa agctactcct gccaagtcac ccatgagggc 600

tcgaccgtcg aaaagaccgt cgccccgaca gagtgttct 639

<210>15

<211>5391

<212>DNA

<213 > mankind

<400>15

ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 60

ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 120

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 180

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 240

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 360

tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 420

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 480

gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 540

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 600

ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 660

caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 720

gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta 780

gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 840

tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 900

acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc tttttctagt 960

agcaactgca actggagtac attcagatat ccagatgacc cagtccccga gctccctgtc 1020

cgcctctgtg ggcgataggg tcaccatcac ctgccgtgcc agtcaggaca tccgtaatta 1080

tttgaactgg tatcaacaga aaccaggaaa agctccgaaa ctactgattt actatacctc 1140

ccgcctggag tctggagtcc cttctcgctt ctctggttct ggttctggga cggattacac 1200

tctgaccatc agtagtctgc aaccggagga cttcgcaact tattactgtc agcaaggtaa 1260

tactctgccg tggacgttcg gacagggcac caaggtggag atcaaacgaa ctgtggctgc 1320

accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt 1380

tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa 1440

cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac 1500

ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta 1560

cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg 1620

agagtgttaa gcttggccgc catggcccaa cttgtttatt gcagcttata atggttacaa 1680

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 1740

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcgatcggg aattaattcg 1800

gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag 1860

gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 1920

cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 1980

ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 2040

tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 2100

cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 2160

agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctgt 2220

taacagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 2280

ccaacttaat cgccttgcag cacatccccc cttcgccagc tggcgtaata gcgaagaggc 2340

ccgcaccgat cgcccttccc aacagttgcg tagcctgaat ggcgaatggc gcctgatgcg 2400

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 2460

tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2520

gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2580

acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2640

agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 2700

ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2760

ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 2820

taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2880

aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 2940

tgctctgatg ccgcatagtt aagccaactc cgctatcgct acgtgactgg gtcatggctg 3000

cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 3060

ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 3120

catcaccgaa acgcgcgagg cagtattctt gaagacgaaa gggcctcgtg atacgcctat 3180

ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 3240

gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 3300

tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 3360

ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 3420

ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 3480

gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 3540

gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgatg 3600

acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3660

actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3720

ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3780

cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 3840

gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgccagcag 3900

caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 3960

aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 4020

ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 4080

tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 4140

ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4200

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 4260

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 4320

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 4380

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4440

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4500

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 4560

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4620

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4680

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4740

cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg 4800

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4860

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 4920

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 4980

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 5040

ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 5100

ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 5160

caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatccagc tggcacgaca 5220

ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc 5280

attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 5340

gcggataaca atttcacaca ggaaacagct atgaccatga ttacgaatta a 5391

<210>16

<211>6135

<212>DNA

<213 > mankind

<400>16

attcgagctc gcccgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc 60

attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 120

tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 180

aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 240

cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 300

taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 360

gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 420

tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 480

tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 540

cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg 600

tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag 660

acaccgggac cgatccagcc tccgcggccg ggaacggtgc attggaacgc ggattccccg 720

tgccaagagt gacgtaagta ccgcctatag agtctatagg cccaccccct tggcttcgtt 780

agaacgcggc tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca 840

ctatagaata acatccactt tgcctttctc tccacaggtg tccactccca ggtccaactg 900

cacctcggtt ctatcgattg aattccacca tgggatggtc atgtatcatc ctttttctag 960

tagcaactgc aactggagta cattcagaag ttcagctggt ggagtctggc ggtggcctgg 1020

tgcagccagg gggctcactc cgtttgtcct gtgcagcttc tggctactcc tttaccggct 1080

acactatgaa ctgggtgcgt caggccccag gtaagggcct ggaatgggtt gcactgatta 1140

atccttataa aggtgttact acctatgccg atagcgtcaa gggccgtttc actataagcg 1200

tagataaatc caaaaacaca gcctacctgc aaatgaacag cctgcgtgct gaggacactg 1260

ccgtctatta ttgtgctaga agcggatact acggcgatag cgactggtat tttgacgtct 1320

ggggtcaagg aaccctggtc accgtctcct cggcctccac caagggccca tcggtcttcc 1380

ccctggcacc ctcctccaag agcacctctg ggggcacagc ggccctgggc tgcctggtca 1440

aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg accagcggcg 1500

tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc agcgtggtga 1560

ctgtgccctc tagcagcttg ggcacccaga cctacatctg caacgtgaat cacaagccca 1620

gcaacaccaa ggtggacaag aaagttgagc ccaaatcttg tgacaaaact cacacatgcc 1680

caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc cccccaaaac 1740

ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga 1800

gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg 1860

ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca 1920

ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag 1980

ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac 2040

aggtgtacac cctgccccca tcccgggaag agatgaccaa gaaccaggtc agcctgacct 2100

gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc 2160

cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttcctct 2220

acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg 2280

tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta 2340

aatgagtgcg acggccctag agtcgacctg cagaagcttg gccgccatgg cccaacttgt 2400

ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 2460

catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 2520

tctggatcga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga 2580

ggaacttggt taggtacctt ctgaggcgga aagaaccatc tgtggaatgt gtgtcagtta 2640

gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2700

tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 2760

atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta 2820

actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 2880

gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 2940

ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc 3000

gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccccttcg 3060

ccagttggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgtagcc 3120

tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3180

accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 3240

tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 3300

cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 3360

ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 3420

tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 3480

gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 3540

tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 3600

aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 3660

tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc aactccgcta 3720

tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc 3780

tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 3840

tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagta ttcttgaaga 3900

cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 3960

tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 4020

taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 4080

tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 4140

gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 4200

gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 4260

cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 4320

tgtggcgcgg tattatcccg tgatgacgcc gggcaagagc aactcggtcg ccgcatacac 4380

tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 4440

atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 4500

ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 4560

gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 4620

gagcgtgaca ccacgatgcc agcagcaatg gcaacaacgt tgcgcaaact attaactggc 4680

gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 4740

gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 4800

gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 4860

cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 4920

atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 4980

tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 5040

ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 5100

gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 5160

tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 5220

ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 5280

ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 5340

gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 5400

ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 5460

tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 5520

cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 5580

agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 5640

agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 5700

gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 5760

tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 5820

accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 5880

gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 5940

attcattaat ccaactggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 6000

gcaattaatg tgagttacct cactcattag gcaccccagg ctttacactt tatgcttccg 6060

gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 6120

catgattacg aatta 6135

<210>17

<211>1353

<212>DNA

<213 > mankind

<400>17

gaagttcagc tggtgcagtc tgggggaggt gtggaacggc cgggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatgcca tgagctgggt ccgccaagct 120

ccagggaagg ggctggagtg ggtctctggt atcaattggc agggtggtag cacaggatat 180

gcagactctg tgaagggccg agtcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaatcctg 300

ggtgccggac ggggctggta cttcgattac tgggggaagg ggaccacggt caccgtctcg 360

agtgcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtggtg actgtgccct ctagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggaa 1080

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccgggt aaa 1353

<210>18

<211>451

<212>PRT

<213 > artificial sequence

<220>

<223 > composition sequence

<400>18

G1u Val Gln Leu Val Gln Ser Gly Gly Gly Val Glu Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asn Trp Gln Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ile Leu Gly Ala Gly Arg Gly Trp Tyr Phe Asp Tyr Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>19

<211>639

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<400>19

tctgagctga ctcaggaccc tgctgtgtct gtggccttgg gacagacagt caggatcaca 60

tgctcaggag acagcctcag aagctattat gcaagctggt accagcagaa gccaggacag 120

gcccctgtac ttgtcatcta tggtgcaaac aacaggcctt cagggatccc agaccgattc 180

tctggctcca gctcaggaaa cacagcttcc ttgaccatca ctggggctca ggcggaagat 240

gaggctgact attactgtaa ctccgcggac agcagtggta accatgtggt attcggcgga 300

gggaccaagc tgaccgtcct tggccaacct aaggctgcac catctgtcac cctcttcccg 360

ccatcttctg aggagttgca agctaacaaa gccactcttg tgtgcctgat cagtgacttc 420

tatcccggag cggtcacagt agcgtggaag gcggatagct cccccgtaaa ggctggcgtc 480

gagacgacta ccccttcgaa gcagagcaac aacaaatacg ccgccagcag ctacctgtcg 540

ctgaccccag aacagtggaa gagccacaaa agctactcct gccaagtcac ccatgagggc 600

tcgaccgtcg aaaagaccgt cgccccgaca gagtgttct 639

<210>20

<211>213

<212>PRT

<213 > artificial sequence

<220>

<223 > composition sequence

<400>20

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr

1 5 10 15

Val Arg Ile Thr Cys Ser Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly

35 40 45

Ala Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser

50 55 60

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Asn Ser Ala Asp Ser Ser Gly Asn His Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys Ser

210

<210>21

<211>48

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<400>21

catgccaagg agactaactc agataatatt aagctagctg gtaccagc 48

<210>22

<211>48

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<220>

<221>mi sc_feature

<222>15，16，24，25，30，31

<223 > n=A, T, C and G

<220>

<221>mi sc_feature

<222>17，26，32

<223 > s=G and C

<400>22

catgccaagg agacnnsctc agannstatn nsgctagctg gtaccagc 48

<210>23

<211>44

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<400>23

gtcatctatg gtaaataata acggccgtct ggcatcccag accg 44

<210>24

<211>47

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<220>

<221>mi sc_feature

<222>19，20，22，23，25，26

<223 > n=A, T, C or G

<220>

<221>mi sc_feature

<222>21，24，27

<223 > s=G and C

<400>24

cttgtcatct atggtaaann snnsnnsccg tctggcatcc cagaccg 47

<210>25

<211>59

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<400>25

gctgactatt actgtaactc ccggtaataa taaggctaac atgtggtatt cggcggagg 59

<210>26

<211>59

<212>DNA

<213 > artificial sequence

<220>

<223 > composition sequence

<220>

<221>mi sc_feature

<222>25，26，28，29，31，32，37，38

<223 > n=A, T, C or G

<220>

<221>mi sc feature

<222>27，30，33，39

<223 > s=G and C

<400>26

gctgactatt actgtaactc ccggnnsnns nnsggcnnsc atgtggtatt cggcggagg 59

<210>27

<211>11

<212>PRT

<213 > artificial sequence

<220>

<223 > composition sequence

<400>27

Asn Ser Arg Asp Ser Ser Gly Ser His Val Val

1 5 10

<210>28

<211>11

<212>PRT

<213 > artificial sequence

<220>

<223 > composition sequence

<400>28

Asn Ser Arg Ser Tyr Ser Gly Asn His Val Val

1 5 10

<210>29

<211>11

<212>PRT

<213 > artificial sequence

<220>

<223 > composition sequence

<400>29

Asn Ser Arg Ser Ser Ser Gly Ser His Val Val

1 5 10

Claims (52)

1. Anti-DR5 antibody or its fragment, it comprises sudden change in the heavy chain of full length antibody 16E2 and light chain at SEQ ID NO:11 and 13 shown in respectively, wherein said antibody or antibody fragment have the affinity to DR5 higher than full length antibody 16E2 and can activate in cancerous cell or stimulate apoptosis, and described sudden change comprises

Be selected from one group of heavy chain sudden change of lower group in aminoacid sequence SEQ ID NO:11: (i) N53Q, L102Y; (ii) M34L, N53Q, L102Y; (iii) N53Y, L102Y; (iv) M34L, N53Y, L102Y; (v) G33A, N53Q, L102Y; (vi) M34L, N53Y, L102Y; (vii) G33A, N53Y, L102Y; (viii) T28A, N53Q, L102Y; (ix) T28A, N53Y, L102Y; And the one group of light chain that is selected from lower group in aminoacid sequence SEQ ID NO:13 suddenlys change: (i) Q24S, G50K, K51D, N52S, N53E, H95bY; (ii) Q24S, K51A, D92S, S93Y; (iii) Q24S, K51A, R91A.

2. the Anti-DR5 antibody of claim 1 or its fragment, wherein said antibody or antibody fragment with full length antibody 16E2 in conjunction with identical epi-position.

3. the Anti-DR5 antibody of claim 1 or its fragment, it also comprises the sudden change of one or many places in total length 16E2 antibody heavy chain variable region framework shown in aminoacid sequence SEQ ID NO:11.

4. the Anti-DR5 antibody of claim 3 or its fragment, wherein said framework sudden change is selected from lower group: Q6E, V11L, E12V, R13Q and K105Q.

5. the Anti-DR5 antibody of claim 4 or its fragment, it comprises all frameworks sudden change Q6E, V11L, E12V, R13Q and K105Q.

6. the Anti-DR5 antibody of claim 1-5 any one or its fragment, it comprises at least one sudden change G99A of place and R100A in aminoacid sequence SEQ ID NO:11.

7. the Anti-DR5 antibody of claim 1 or its fragment, it comprises following sudden change: the G33A in sequence SEQ ID NO:11, N53Q, the Q24S in L102Y and sequence SEQ ID NO:13, K51A, R91A.

8. the Anti-DR5 antibody of claim 1 or its fragment, it comprises following sudden change: the G33A in sequence SEQ ID NO:11, N53Y, the Q24S in L102Y and sequence SEQ ID NO:13, K51A, R91A.

9. the Anti-DR5 antibody of claim 1-8 any one or antibody fragment, wherein said fragment is selected from lower group: Fab, Fab ', F (ab ') 2 and Fv fragment, double antibody, single-chain antibody molecule and the multi-specificity antibody formed by antibody fragment.

10. the Anti-DR5 antibody of claim 9 or antibody fragment, wherein said antibody is single-chain antibody.

11. the Anti-DR5 antibody of claim 9 or antibody fragment, wherein said fragment is the Fv fragment.

12. the Anti-DR5 antibody of claim 1-11 any one or antibody fragment, wherein said antibody has the active anticancer higher than full length antibody 16E2.

13. the Anti-DR5 antibody of claim 1-12 any one or antibody fragment, wherein said antibody has the effect higher than full length antibody 16E2, and this effect is measured in the tumor-killing algoscopy in vitro.

14. the Anti-DR5 antibody of claim 1-13 any one or antibody fragment, it is chimeric, humanized or people's antibody.

15. the Anti-DR5 antibody of claim 1-14 any one or antibody fragment, the cytotoxicity of its mediate antibody dependent cell (ADCC).

16. the Anti-DR5 antibody of claim 1-15 any one or its fragment, it is dimeric form.

17. the Anti-DR5 antibody of claim 1-16 any one or its fragment, itself and anti-human IgG Fc district are crosslinked.

18. the Anti-DR5 antibody of claim 1-17 any one or antibody fragment, itself and epitope tag sequence merge.

19. chimeric molecule, the antibody that it comprises the claim 1-18 any one merged with the allogeneic amino acid sequence.

20. the chimeric molecule of claim 19, wherein said allogeneic amino acid sequence comprises immunoglobulin sequences.

21. the chimeric molecule of claim 20, wherein said immunoglobulin sequences is anti-human IgG Fc district.

22. the Anti-DR5 antibody of claim 1-18 any one or antibody fragment, it is used for the treatment of.

23. the Anti-DR5 antibody of claim 22 or antibody fragment, it is used for the treatment of cancer.

24. the Anti-DR5 antibody of claim 23 or antibody fragment, wherein said cancer is selected from lower group: cancer, lymphoma, blastoma, sarcoma and leukemia.

25. the Anti-DR5 antibody of claim 23 or antibody fragment, wherein said cancer is selected from lower group: squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer (NSCLC), non_hodgkin lymphoma, blastoma, human primary gastrointestinal cancers, renal carcinoma (renal cancer), ovarian cancer, liver cancer (liver cancer), gastric cancer, bladder cancer, hepatoma (hepatoma), breast carcinoma, colon cancer, colorectal carcinoma, cancer of pancreas, carcinoma of endometrium, salivary-gland carcinoma, renal carcinoma (kidney cancer), carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, hepatocarcinoma (hepatic carcinoma), and head and neck cancer.

26. the Anti-DR5 antibody of claim 25 or antibody fragment, wherein said cancer is NSCLC, non_hodgkin lymphoma, colorectal carcinoma or cancer of pancreas.

27. the Anti-DR5 antibody of claim 24 or antibody fragment, wherein said cancer is adenocarcinoma.

28. the Anti-DR5 antibody of claim 27 or antibody fragment, wherein said adenocarcinoma is colorectal carcinoma, cancer of pancreas or adenocarcinoma metastatic.

29. the nucleic acid separated, the Anti-DR5 antibody of its coding claim 1-18 any one or heavy chain or the light chain of its fragment.

30. carrier, the nucleic acid that it comprises claim 29.

31. host cell, the nucleic acid that it comprises claim 29.

32. the method for the Anti-DR5 antibody of production claim 1-18 any one, be included in the host cell of cultivating claim 31 under the condition that DNA expresses.

33. be used for the treatment of the compositions of cancer, the Anti-DR5 antibody that it comprises claim 1-18 any one or its fragment, and carrier, wherein said cancer is colorectal carcinoma, lymphoma, cancer of pancreas or pulmonary carcinoma.

34. the compositions of claim 33, wherein said carrier is pharmaceutically acceptable carrier.

35. the compositions of claim 34, also comprise other anticarcinogen.

36. the compositions of claim 35, wherein said other anticarcinogen is antibody.

37. the compositions of claim 36, wherein said antibody is selected from lower group: other Anti-DR5 antibody, Rituxan (rituximab) and VEGF antibody.

38. the compositions of claim 35, wherein said other anticarcinogen is chemotherapeutics.

39. the compositions of claim 38, wherein said chemotherapeutics is selected from lower group: CPT-11 (irinotecan), gemcitabine, carboplatin, taxol and paclitaxel.

40. the compositions of claim 35, wherein said other anticarcinogen is the Apo2L part of the amino acid/11 14-281 that comprises SEQ ID NO:1.

41. the Anti-DR5 antibody of claim 1-18 any one or the purposes of its fragment in the medicine for the preparation of apoptosis-induced, described inducing comprises Anti-DR5 antibody or its fragment that makes mammalian cancer cells be exposed to claim 1-18 any one.

42. the purposes of claim 41, wherein make described mammalian cancer cells be exposed to the medicament of activation DR5.

43. the Anti-DR5 antibody of claim 1-18 any one or its fragment purposes in the medicine for the preparation of the treatment cancer, described treatment comprises Anti-DR5 antibody or its fragment of mammalian subject being used to the claim 1-18 any one of effective dose, and wherein said cancer is colorectal carcinoma, lymphoma, cancer of pancreas or pulmonary carcinoma.

44. the purposes of claim 43, wherein said mammalian subject is the human experimenter.

45. the purposes of claim 43 or 44, wherein said cancer is selected from lower group: squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer (NSCLC), non_hodgkin lymphoma, blastoma, colon cancer, colorectal carcinoma and cancer of pancreas.

46. the purposes of claim 45, wherein said cancer is NSCLC, colorectal carcinoma, non_hodgkin lymphoma or cancer of pancreas.

47. the purposes of claim 43 or 44, wherein said cancer is adenocarcinoma.

48. the purposes of claim 47, wherein said adenocarcinoma is colorectal carcinoma, cancer of pancreas or adenocarcinoma metastatic.

49. the purposes of claim 43-48 any one, also comprise and use other anticarcinogen.

50. be used for the treatment of the test kit of cancer, comprise container and be contained in the compositions in described container, the Anti-DR5 antibody that wherein said compositions comprises claim 1-18 any one or its fragment, wherein said cancer is colorectal carcinoma, lymphoma, cancer of pancreas or pulmonary carcinoma.

51. the test kit of claim 50, also be included in external or use in vivo the description of Anti-DR5 antibody.

52. the test kit of claim 51, the treatment of wherein said description explanation to cancer.

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